Method to provide bacterial ghosts provided with antigens

ABSTRACT

Methods for improving binding of a proteinaceous substance to cell-wall material of a Gram-positive bacterium are disclosed. The proteinaceous substance includes an AcmA cell-wall binding domain, homolog or functional derivative thereof. The method includes treating the cell-wall material with a solution capable of removing a cell-wall component such as a protein, lipoteichoic acid or carbohydrate from the cell-wall material and contacting the proteinaceous substance with the cell-wall material.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of International ApplicationNumber PCT/NL02/00383 filed on Jun. 11, 2002, designating the UnitedStates of America, not yet published, the contents of the entirety ofwhich are incorporated by reference.

TECHNICAL FIELD

[0002] The present invention pertains to a method for obtainingcell-wall material of Gram-positive bacteria with an improved capacityfor binding a proteinaceous substance comprising an AcmA cell-wallbinding domain, as well as pharmaceutical compositions including theobtained cell-wall material.

BACKGROUND

[0003] Heterologous surface display of proteins (Stahl and Uhlen,TIBTECH May 1997, 15, 185-192) on recombinant microorganisms via thetargeting and anchoring of heterologous proteins to the outer surface orthe cell wall of host cells, such as yeast, fungi, mammalian cells,plant cells, and bacteria, has been possible for several years. Displayof heterologous proteins at the surface of these cells has taken manyforms including the expression of reactive groups such as antigenicdeterminants, heterologous enzymes, single-chain antibodies,polyhistidyl tags, peptides, and other compounds. Heterologous surfacedisplay has been applied as a tool for applied and fundamental researchin microbiology, molecular biology, vaccinology and biotechnology.Another application of bacterial surface display has been thedevelopment of live-bacterial-vaccine delivery systems. The cell-surfacedisplay of heterologous antigenic determinants has been consideredadvantageous for inducing antigen-specific immune responses in liverecombinant cells used for immunization. Another application has been-the use of bacterial surface display in generating whole-cellbioadsorbents or biofilters for environmental purposes,microbiocatalysts, and diagnostic tools.

[0004] Generally, chimeric proteins include an anchoring or targetingportion that is specific and selective for the recombinant organism,wherein the anchoring portion is combined with the reactive group, suchas the antigenic determinant, heterologous enzyme, single-chainantibody, polyhistidyl tag, peptide, or other compound. A well knownanchoring portion comprises the so-called LPXTG (SEQ ID NO: 1) box,which covalently binds to a Staphylococcus bacterial surface, i.e., inthe form of a fully integrated membrane protein. In this manner, atleast two polypeptides of different genetic origins may be joined by anormal peptide bond to produce a chimeric protein. For example, PCTInternational Patent Publication No. WO 94/18330, which relates to theisolation of compounds from complex mixtures and the preparation ofimmobilized ligands (bioadsorbents), discloses a method for obtaining aligand comprising anchoring a binding protein in or at the exterior of acell wall of a recombinant cell. The binding protein is essentially achimeric protein produced by the recombinant cell and includes anN-terminal part derived from an antibody that is capable of binding to aspecific compound, wherein the N-terminal part is joined to a C-terminalanchoring part, derived from an anchoring protein purposely selected forbeing functional in the specific recombinant cell chosen. PCTInternational Patent Publication No. WO 97/08553 discloses a method forselectively targeting proteins to the cell wall of Staphylococcus sp.,using anchoring proteins which include long stretches of at least 80-90amino acid long amino acid cell-wall-targeting signals. The signals arederived from the lysostaphin gene or amidase gene of Staphylococcus andencode for proteins that selectively bind to Staphylococcus cell-wallcomponents.

[0005] Vaccine delivery or immunization systems with attenuatedbacterial vector strains that express distinct antigenic determinantsagainst a wide variety of diseases are currently being developed.Mucosal vaccines for nasal or oral passages using these attenuatedbacterial vectors have received a great deal of attention. For example,both systemic and mucosal antibody responses against an antigenicdeterminant of hornet venom have been detected in mice orally colonizedwith a genetically engineered human oral commensal Streptococcusgordonii strain that expresses the hornet venom antigenic determinant onits surface (Medaglini et al., PNAS 1995, 2; 6868-6872). A protectiveimmune response was also elicited by oral delivery of a recombinantbacterial vaccine that included tetanus toxin fragment C constitutivelyexpressed in Lactococcus lactis (Robinson et al., Nature Biotechnology1997, 15; 653-657). Mucosal immunization is considered an effectivemeans of inducing IgG and secretory IgA antibodies directed againstspecific pathogens of mucosal surfaces.

[0006] Immunogens expressed by bacterial vectors may be presented in aparticulate form to antigen-presenting cells, such as M-cells, of theimmune system and therefore should be less likely to induce tolerancewhen compared to soluble antigens. Additionally, the existence of acommon mucosal immune system permits immunization of one specificmucosal surface in order to induce secretion of antigen-specific IgA andother specific immune responses at distant mucosal sites. A drawback tousing bacterial vectors for immunization is the potential of thebacterial strain causing inflammation or disease and potentially leadingto fever or bacteremia. Instead of using attenuated bacterial strainsthat may become pathogenic, recombinant commensal bacteria, such asStreptococcus sp. or Lactococcus sp., may be used as vaccine carriers.

[0007] A potential problem with recombinant commensal microorganisms isthat they may colonize the mucosal surfaces and generate a long termexposure to the target antigens expressed and released by therecombinant microorganisms which may cause immune tolerance.

[0008] Additionally, the use of genetically modified microorganisms thatcontain recombinant nucleic acid has met considerable opposition fromthe public as a whole, stemming from a low level acceptance of productswhich contain recombinant DNA or RNA. Similar objections exist againsteven the use of attenuated pathogenic strains or against proteins, orparts of proteins, derived from pathogenic strains. Further, theheterologous surface display of proteins described herein entails theuse of anchoring or targeting proteins specific and selective for alimited set of microorganisms, which are of recombinant or pathogenicnature which greatly restricts their potential applications.

[0009] The protein anchor of L. lactis, AcmA (cA), its homologs andfunctional derivatives (PCT International Patent Publication No.W099/25836) bind in a non-covalent manner to a wide variety ofGram-positive bacteria. Binding also occurs to isolated cell-wallmaterial. The ligand to which the protein anchor of L. lactis binds inthese cell walls is currently unknown.

[0010] The use of a gram-positive, food-grade bacterium, such asLactococcus lactis, offers significant advantages over the use of otherbacteria, such as Salmonella, as a vaccine delivery vehicle. Forinstance, L. lactis does not replicate in or invade human tissues andreportedly possesses low intrinsic immunity (Norton et al. 1994).Further, mucosal delivered L. lactis that expresses tetanus toxinfragment C has been shown to induce antibodies that protect mice againsta lethal challenge with tetanus toxin even if the carrier bacteria waskilled prior to administration (Robinson et al. 1997). The killedbacteria still contain recombinant DNA that will be spread into theenvironment, especially when used in wide-scale oral-immunizationprograms. However, the uncontrollable shedding of recombinant DNA intothe environment may have the risk of being taken up by other bacteria orother microorganisms.

SUMMARY OF THE INVENTION

[0011] The invention discloses a method for improving binding of aproteinaceous substance to cell-wall material of a Gram-positivebacterium. The proteinaceous substance comprises at least one repeat,but may comprise two or three repeat sequences of an AcmA cell-wallbinding domain, homolog or functional derivative thereof. The methodcomprises treating the cell-wall material with a solution capable ofremoving a cell-wall component, such as a protein, lipoteichoic acid orcarbohydrate, from the cell-wall material and contacting theproteinaceous substance with the treated cell-wall material. Improvedbinding may be obtained by treating the cell-wall material with asolution capable of removing a cell-wall component. The cell-wallmaterial may be subsequently stored until it is contacted with a desiredfusion protein. The fusion protein may comprise an AcmA cell-wallbinding domain, homolog or functional derivative thereof where thecell-wall material is contacted with the fusion protein. The method ofthe present invention may be used to obtain cell-wall material with animproved capacity for binding a proteinaceous substance comprising theAcmA cell-wall binding domain, homolog or functional derivative thereof.

[0012] The invention also discloses a method for removing componentsfrom a bacterial cell-wall comprising treating whole cells with asolution capable of removing a cell-wall component such as a protein,lipoteichoic acid or carbohydrate from the cell-wall material. Thecell-wall material obtained by the present invention yields cell-wallmaterial with at least 20%, better 30%, best 40% or even 50% ofrelatively empty, but intact, cell envelopes which include inertspherical microparticles. The inert spherical microparticles will bereferred to herein as bacterial “ghosts.” The tenn “ghosts” reflects thesize and shape of the bacterium from which the ghosts are obtained.

[0013] The invention also discloses a method for obtaining cell-wallmaterial of a Gram-positive bacterium with an improved capacity forbinding with a proteinaceous substance comprising an AcmA cell-wallbinding domain, homolog or functional derivative thereof The methodcomprises treating the cell-wall material with a solution capable ofremoving a cell-wall component such as a protein, lipoteichoic acid orcarbohydrate from the cell-wall material, wherein the cell-wall materialcomprises spherical peptidoglycan microparticles referred to herein asghosts.

[0014] Methods to extract bacterial cell-wall material with a solutionhave been described in EP 0 545 352 A and Brown et al. (Prep. Biochem.6:479, 1976). A method to obtain purified soluble peptidoglycan frombacteria by exposure to TCA has been disclosed. The cited referencesdescribe procedures in which cells are mechanically disrupted, whereinthe resulting cell fragments are treated with TCA to extractpeptidoglycans from the cell-wall. The cited methods provide apeptidoglycan preparation and a lysed, randomly fragmented cell-wallpreparation from which cell-wall components have been removed. However,these methods do not yield ghosts. Furthermore, the methods do not allowtargeting with a proteinaceous substance comprising an AcmA cell-wallbinding domain, homolog or functional derivative thereof.

[0015] The method of the present invention is aimed at yielding ghostsfrom which cell-wall components have been removed. The use of ghosts fordisplay of proteinaceous substances has advantages over the use of thedisrupted cell--wall material. For example, binding the proteinaceoussubstance to bacterial ghosts results in a higher packing density whencompared to binding a substance to mechanically disrupted cell-wallmaterial. A high density surface display of proteins is favorable forapplication in industrial processes. In one embodiment, the presentinvention discloses a method for obtaining the cell-wall material notinvolving rupture.

[0016] Cell-wall material obtained by mechanical disruption methodssuffers from several practical drawbacks. Because cells are completelybroken with mechanical disruption, intracellular materials are releasedfrom the cell and cell-wall fragments need to be separated from acomplex mixture of proteins, nucleic acids, and other cellularcomponents. The released nucleic acids may increase the viscosity of thesolution and complicate processing steps, especially chromatography. Thecell debris produced by mechanical lysis also often includes small cellfragments which are difficult to remove. These problems are overcomewhen ghosts are produced using methods of the present invention. Theuniform composition of a ghost preparation including particle size andshape offers other advantages for subsequent purification and isolationsteps. The invention thus discloses a method of obtaining cell-wallmaterial not involving rupture of the cell-wall, wherein the resultingcell-wall material comprises ghosts.

[0017] The use of bacterial ghosts is often preferable when compared tothe use of mechanically disrupted cell-wall bacteria for the surfacedisplay of immunogenic determinants. In contrast to mechanicaldisruption procedures, ghosts are produced by a process that preservesmost of the bacteria's native spherical structure. Bacterial ghosts arebetter able to bind to and/or are more easily taken up by specific cellsor tissues than mechanically disrupted cell-wall material. The abilityof bacterial ghosts to target macrophages or dendritic cells enhancestheir functional efficacy. Thus, the non-recombinant, non-living ghostsystem disclosed by the present invention is well suited as a vaccinedelivery vehicle. Accordingly, the invention discloses a method forobtaining ghosts, wherein the ghosts have an improved capacity forbinding with a proteinaceous substance and have an enhanced induction ofthe cellular immune response.

[0018] The invention also discloses a method for binding a proteinaceoussubstance to the cell-wall material of a Gram-positive bacterium,wherein the proteinaceous substance comprises an AcmA cell-wall bindingdomain, homolog or functional derivative thereof. The method comprisestreating the cell-wall material with a solution capable of removing acell-wall component such as a protein, lipoteichoic acid or carbohydratefrom the cell-wall material, and subsequently contacting theproteinaceous substance with the cell-wall material. The cell-wallmaterial comprises ghosts which have been produced by the presentinvention which does not involve rupture of the bacterial cell-wall.

[0019] In another embodiment, the solution capable of removing thecell-wall material has a pH that is lower than the calculated Pi valueof the AcmA cell-wall binding domain, homolog or functional derivativethereof. Particularly, the solution comprises an acid such as aceticacid (HAc), hydrochloric acid (HCl), sulphuric acid (H₂SO₄), trichloroacetic acid (TCA), trifluoro acetic acid (TFA), and monochloro aceticacid (MCA). The concentration of the acid in the solution will bedependent on the desired pH value which may be determined by calculationusing computer program such as DNA star or Clone Manager. For instance,when the calculated pI is >8, pH values of about 6 to 4 may suffice foreffecting appropriate binding. When pI values are calculated to belower, such as around 6, pH values of 3-4 may be selected. When domainswith calculated pI values ranging from 8 to 12 are encountered, usingthe solution comprising 0.06 to 1.2 M TCA, or comparable acid, maysuffice.

[0020] The binding may be improved by heating the cell-wall material orghosts in the solution. However, precise requirements for the heatingmay vary depending on the cell-wall material or ghosts. However, heatingfor 5-25 minutes at approximately boiling temperature (i.e., 100° C.)will often generate the desired cell-wall material with improved bindingcapacity. The cell-wall material may then be washed and pelleted (e.g.,by centrifugation) from the treatment solution and subsequently bestored (e.g., by freezing) or freeze-drying until further use. Suchcell-wall material includes spherical peptidoglycan microparticles thatusually reflect the size and shape of the bacterium from which they wereobtained.

[0021] In one embodiment, the cell-wall material is derived from aLactococcus, a Lactobacillus, a Bacillus or a Mycobacterium sp. The cellwalls of Gram-positive bacteria include complex networks ofpeptidoglycan layers, proteins, lipoteichoic acids and other modifiedcarbohydrates. Generally, chemical treatment of the cell-wall materialmay be used to remove cell-wall components such as proteins,lipoteichoic acids and carbohydrates, wherein the chemical treatmentyields purified peptidoglycan (Morata de Ambrosini et al. 1998). Sodiumdodecyl sulphate (SDS) is also commonly used to remove proteins.Trichloric acid (TCA) is known to specifically remove lipoteichoic acidsand carbohydrates from cell-wall isolates. Phenol, formamide andmixtures of chloroform and methanol are other examples of organicsolvents that may be used to enhance the purification of peptidoglycan.

[0022] In the present invention, the effect of the pretreatment of wholecells of gram-positive bacteria with these and other chemicals inrelation to binding technology provides the possibility to obtainbacterial ghosts or cell-wall material derived from the bacteria whichpossess new traits (i.e., different binding properties) without theintroduction of recombinant DNA.

[0023] In another embodiment, the present invention discloses theincorporation of cell-wall material with improved binding capacity forAcmA-type anchors into a composition, such as pharmaceuticalcomposition, with a proteinaceous substance comprising an AcmA-typeanchor. Reactive groups, such as antigenic determinants, heterologousenzymes, single-chain antibodies, polyhistidyl tags, peptides, and othercompounds may be bound to the cell-wall material as disclosed herein byproviding reactive groups with an AcmA-type anchor, and subsequentlycontacting the cell-wall material with the reactive groups to improvebinding capacity. Other reactive groups include fluorescing protein,luciferase, binding protein or peptide, antibiotics, hormones,non-peptide antigenic determinants, carbohydrates, fatty acids, aromaticsubstances or reporter molecules.

[0024] In another embodiment, the invention discloses the use ofcell-wall material in generating bioadsorbents or biofilters forenvironmental purposes, microbiocatalysts, and diagnostic tools. Forinstance, the use of immobilized biocatalysts, such as enzymes or wholemicrobial cells, has increased steadily during the past decade in thefood, pharmaceutical and chemical industries. The immobilizedbiocatalysts are more stable, easier to handle, and can be usedrepeatedly in industrial processes in comparison to their freecounterparts. Immobilization of enzymes typically requires a chemicalstep to link the enzyme to an insoluble support. However, chemicaltreatments may negatively affect the enzymes. Alternatively, enzymes maybe immobilized by incorporation in gels with the disadvantage-thatdiffusion of the substrate into the gel slows down the process.

[0025] As disclosed herein, large-scale immobilization of enzymaticallyactive proteins may be accomplished by surface displaying proteins ongram-positive cells or cell-wall material. For instance, theimmobilization of a fusion protein comprising α-amylase or β-lactamasefused to the AcmA-protein anchor domain has been demonstrated herein inL. lactis. The addition of the AcmA-anchor fusion protein resulted inthe stable attachment of heterologous proteins to the surface of L.lactis and other gram-positive bacteria. Further, pretreating L. lactiscells and other gram-positive cells with acid as described hereinresults in a high density surface display of heterologous proteins andis a prerequisite for application in industrial processes. Further, thecarrier or gram-positive cells may be obtained in high yield and benon-recombinant. Thus, a method disclosed herein may be used toeconomically produce the immobilized enzyme and make the AcmA-proteinanchor a useful approach for the surface display of enzymes ongram-positive cells.

[0026] Another industrial application of an immobilized enzyme is theisomerization of glucose which is catalyzed by glucose isomerase andused during the production of high-fructose corn syrup. This process maybe made economically feasible by immobilizing the glucose isomerase. Theproductivity of glucose isomerase is improved by increasing thestability of epoxide hydrolase in organic solvents by immobilization tomicrobial cells or cell-wall material as described herein.

[0027] Immobilized enzymes may also be used to treat waste water orindustrial effluent. For instance, industrial effluents containing lowvalue chemicals produced during synthesis of the commodity chemicalsepichlorohydrin and propylene oxide may be treated by using immobilizedhaloalkane dehalogenase to recycle these low value products into themanufacturing process.

[0028] The invention further discloses chimeric or hybrid AcmA-typeanchors for the preparation of a composition that has new bindingproperties. The AcmA-type anchors can be divided into two groups ofhybrids based on their pI (see, table 3). A large group includes hybridswith a pI higher than 8, but lower than 10, and a smaller group includeshybrids with a relatively low pI (i.e., <5). Hybrid AcmA type anchorsare disclosed with at least one AcmA type domain and a relatively highcalculated pI, and another AcmA type domain is disclosed with arelatively lower calculated pI. The resulting hybrid anchor has anintermediate calculated pI which is useful when release of the boundproteinaceous substance at a higher pH is contemplated. Such acomposition may be routed through the stomach, which has relative lowpH, such that the composition releases its anchor bound reactive groupsin the intestines which have a higher pH.

[0029] The invention also discloses a proteinaceous substance comprisingan AcmA cell-wall binding domain, homolog or functional derivativethereof wherein the binding domain is a hybrid of at least two differentAcmA-type cell-wall binding domains, homologs or functional derivativesthereof. The proteinaceous substance may comprise an AcmA cell-wallbinding domain, homolog or functional derivative thereof where thebinding domain is a hybrid of at least two different AcmA repeatsequences and has a calculated pI lower than 10. For instance, a hybridprotein anchor including the A1 and A2 repeat sequences of AcmA and theD1 repeat sequence of AcmD may be constructed. Such a hybrid domain maycomprise at least one AcmA type domain with a relatively high calculatedpI and another AcmA type domain with a relatively lower calculated pI.The domain with the relatively high pI may be derived from, orfunctionally equivalent to, the AcmA type domain of the lactococcalcell-wall hydrolase AcmA. Of course, many other domains with a high pIare known, such as those disclosed in table 3. A domain with arelatively low pI may be derived from, or functionally equivalent to,the AcmA type domain of the lactococcal cell-wall hydrolase AcmD.However, other domains with relatively low pI are known, including thosedisclosed in table 3.

[0030] The invention further discloses a proteinaceous substancecomprising a hybrid domain with at least two stretches of amino acids,wherein each stretch corresponds to a domain repeat sequence and islocated adjacent to each other. The stretches may be separated by one ormore amino acid residues of a short distance, i.e., 3-6 to 10-15 aminoacids apart, by a medium distance, i.e., 15-100 amino acids apart, or bylonger distances, i.e., >100 amino acid residues apart.

[0031] In another embodiment, the invention discloses a proteinaceoussubstance with a hybrid AcmA domain that further comprises a reactivegroup. Reactive groups that may be used include, without limitation,antigenic determinants, heterologous enzymes, single-chain antibodies orfragments thereof, polyhistidyl tags, fluorescing proteins, luciferase,binding proteins or peptides, antibiotics, hormones, non-peptideantigenic determinants, carbohydrates, fatty acids, aromatic substances,inorganic particles such as latex, and reporter molecules. The reactivegroup may also include AcmA cell-wall binding domains, homologs orfunctional derivatives thereof wherein the binding domain is a hybrid ofat least two different AcmA cell-wall binding domains, homologs orfunctional derivatives thereof that are useful in heterologous surfacedisplay and are broadly reactive with cell-wall components of a broadrange of micro-organisms. As used herein, the AcmA cell-wall bindingdomains, homologs and functional derivitives thereof will also bereferred to as hybrid AcmA domains.

[0032] The invention further discloses reactive groups which arenon-protein moieties, including substances such as antibiotics,hormones, aromatic substances, inorganic particles, or reportermolecules. The substances may be constructed by binding an antibiotic,such as penicillin, tetracycline or various other antibiotics, ahormone, such as a steroid hormone, or any other compound to a bindingdomain produced by the present invention. Such binding may be achievedusing various techniques known in the art and may function to label or“flag” the binding domain. For instance, a binding domain may be boundto a reporter molecule such as fluorescent nanoparticles, i.e., FITC orHRPO, wherein tools are generated that may be used in diagnostic assaysto detect microorganisms possessing peptidoglycan. Similarly, a bindingdomain may be bound to an antibiotic and used for in vivo parenteraladministration into the bloodstream of humans or animals, or used invitro to bind microorganisms with peptidoglycan in order to increase theconcentration of the antibiotic around the microorganism which may bekilled by the antibiotics.

[0033] The invention further discloses a reactive group which is aprotein moiety which may include, without limitation, antigenicdeterminants, enzymes, single-chain antibodies or fragments thereof,polyhistidyl tags, fluorescing proteins, binding proteins or peptides.For instance, a protein including a reactive group which is anotherprotein or polypeptide is disclosed. The invention also discloses anucleic acid molecule encoding the protein produced using the methods ofthe invention. Such a nucleic acid molecule, comprising single-strandedor double-stranded DNA, RNA or DNA-RNA duplex, comprises nucleic acidsequences which encode a hybrid binding domain. The nucleic acidmolecule may also comprise nucleic acid sequences encoding the reactivegroup polypeptide and may further comprise other nucleic acid sequencesencoding a signal peptide comprising promoter sequences or regulatorynucleic acid sequences.

[0034] A vector comprising a nucleic acid molecule encoding aproteinaceous substance provided by the invention is also disclosed.Examples of vectors include, without limitation, a plasmid, a phage or avirus, wherein the vectors may be constructed using nucleic acids of theinvention and routine skills known in the art. Viral vectors includebaculovirus vectors or comparable vector viruses through which a proteinproduced by the present invention may be expressed or produced in cells,such as insect cells.

[0035] A host cell or expression system including a nucleic acidmolecule or a vector produced using methods of the present invention arealso disclosed. The host cell expressing a protein of the presentinvention may be a microorganism to which the protein is attached. Thehost cell, or expression system, may be a Gram-positive bacterium, aGram-negative bacterium, a yeast cell, an insect cell, a plant cell, amammalian cell, or a cell-free expression system, such as a reticulocytelysate. The host cell or expression system may be constructed orobtained using a nucleic acid or vector of the present invention androutine skills known in the art.

[0036] In a further embodiment, the invention discloses a pharmaceuticalcomposition comprising cell-wall material with an improved bindingcapacity with an immunogen bound thereto which may be useful forvaccination purposes, i.e., a vaccine. The vaccine may be used to invokeimmunity against pathogens, such as malaria, which undergo life cyclestages where the pathogen is not in the blood, but hides in cells.

[0037] The vaccines may be delivered to mucosal surfaces instead ofbeing injected since mucosal surface vaccines are easier and safer toadminister. A L. lactis derived cell-wall material may be used formucosal vaccination since this bacterium is of intestinal origin and noadverse immune reactions are generally expected from L. lactis.

[0038] The vaccine of the invention may also be administered byinjection. When the vaccine is administered through injection, cell-wallmaterial may be derived from a Mycobacterium sp. since mycobacterialcell-wall preparations have beneficial adjuvant properties. Themycobacterial cell-wall vaccine may be mixed with the proteinaceoussubstance carrying the immunogenic determinants used in the vaccine.

[0039] A vaccine produced using a method of the present invention willlikely have a reduced risk of generating undesirable immune responsesagainst cell-wall compounds of unwanted immunogens because the unwantedimmunogens are not included in the vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040]FIG. 1. Schematic map of plasmid pNG3041 that encodes the reporterprotein MSA2::cA that is secreted as a proprotein using the lactococcalPrtP signal- and prosequences (PrtP.sspro). Pnis represents the nisininducible promoter of the nisA gene. T represents the transcriptionalterminator. CmR is the chloramphenicol resistance gene. repC and repAare genes involved in the replication of the plasmid.

[0041]FIG. 2. Fluorescence microscopic images of bacterial cells withexternally bound MSA2::cA. A. Lb. curvatis, Lb. sake and L. lactis cellsthat were not pre-treated prior to binding. B. L. lactis cells that wereTCA pretreated prior to binding. The light colored areas indicate theposition were the reporter protein MSA2::cA binds. The differencebetween L. lactis cells that were not pretreated with TCA (in A) andthose there were TCA pretreated is apparent (in B).

[0042]FIG. 3. Western blots of chemically pretreated L. lactis cellsthat were washed after the pretreatment and incubated with MSA2::cA toallow binding. Unbound MSA2::cA was removed by washing. The drawingshows the MSA2::cA that was bound to the chemically pretreated cells anddetected using an antibody specific for MSA2. The differentpretreatments are indicated above the lanes. MSA2::cA is produced by theproducer cells as a proprotein, pro-MSA2::cA. Some pro-MSA2::cA ispresent in the medium used for binding and binds as indicated by thearrow. A membrane bound protease, HtrA, of the producer cells cleavesoff the pro-sequence resulting in mature MSA2::cA, which also binds tothe pretreated cells as indicated by the asterisk. HtrA also cleaves offthe repeats of the cA anchor. Since there are three repeats, MSA2proteins of several sizes are present in the medium of the producer. Aslong as more than one repeat is present, binding can still occur. Thedouble asterisks point to MSA2::cA from which one or two repeats havebeen cleaved. M is a molecular weight marker. The molecular weights areindicated in the left margin. The two blots have different signalintensities. As a reference, both blots contain the same TCA-pretreatedsamples. The difference in signal intensity is due to differences instain developing time. It is apparent that the TCA and other acidpretreatments produce pronounced effects on the subsequent binding ofMSA2::cA. The conclusions for all chemical pre-treatments are summarizedin Table 1.

[0043]FIG. 4. Coomassie stained SDS-PAGE gel with chemically pre-treatedL. lactis cells. Pre-treatments: (1) no-treatment; (2) HCl; (3) H₂SO₄;(4) HAc; (5) TFA; and (6) TCA. It is apparent that treatment of thecells with HCl, H₂SO₄, TFA or TCA significantly removes an amount ofprotein from the cells.

[0044]FIG. 5. Western blot of L. lactis cells TCA pre-treated withdifferent TCA concentrations and externally bound with MSA2::cA. Arrowand asterisks: as in FIG. 3. Pretreatments: (1) no TCA-treatment; (2) 1%TCA; (3) 5% TCA; (4) 10% TCA; and (5) 20% TCA. An increase in thebinding of MSA2::cA is shown to correlate with increasing amounts of TCAused in the pretreatment.

[0045]FIG. 6. Alignment of cA repeats with cD repeats. AcmA (A1) (SEQ IDNO: 16) is aligned to AcmD (D1) (SEQ ID NO: 19). AcmA (A2) (SEQ ID NO:17) is aligned to AcmD (D2) (SEQ ID NO: 20). AcmA (A3) (SEQ ID NO: 18)is aligned to AcmD (D3) (SEQ ID NO: 21). Consensus repeats SEQ ID NO:163, 164 and 165 are aligned. The amino acids that are in agreement withthe consensus sequence are shown at the bottom of the figure (defined inPCT Publication WO99/25836) are underlined. The asterisks indicateresidues that are identical between the compared repeats.

[0046]FIG. 7. Binding of various anchor-fusion proteins to L. Lactiswith and without TCA pretreatment. Multiple bands shown in one lane arecaused by the different processed forms of MSA2 fusions. Lanes: (1)non-pretreated L. lactis+MSA2::cA; (2) non-treated L. Iactis+MSA2::cD;(3) non-pretreated L. lactis+MSA2; (4) TCA-pretreated L.lactis+MSA2::cA; (5) TCA-pretreated L. lactis+MSA2::cD; and (6)TCA-pretreated L. lactis+MSA2. The effect of TCA pretreatment on thebinding of MSA2::cA is shown (i.e., compare lanes 1 and 4). A minorimprovement for MSA2::cD and no improvement for MSA2 without anchor isobserved. Since there is a signal for MSA2 without the anchor means thatMSA2 by itself has a weak affinity for bacterial cell walls. However,MSA2::cD or MSA2 binding to the pretreated cells cannot be detectedusing fluorescence or electron microscopy (see text). The difference inresults is probably due to a difference in sensitivity of the techniques

[0047]FIG. 8. Fluorescence microscopy image of TCA-pretreated L. lactiscells incubated with MSA2::cA or MSA2::cD. Light colored areas indicatethe position were the reporter fusion protein binds. It appears thatbinding only occurred with MSA2::cA and not with MSA2::cD.

[0048]FIG. 9. Electron microscopy images of L. lactis cells incubatedwith different MSA2 constructs. The black dots represent the position ofbound MSA2 fusion protein. Image A depicts non-pretreated cellsincubated with MSA2::cA. Image B depicts TCA-pretreated cells incubatedwith MSA2::cA. Image C depicts TCA-pretreated cells incubated withMSA2::cD. Image D TCA-pretreated cells incubated with MSA2. Significantbinding, shown by black dots, is only visible in the TCA-pretreatedcells incubated with MSA2::cA (B).

[0049]FIG. 10. Binding of different anchor-fusion proteins to B.subtilis with and without TCA pretreatment. The drawing is a Westernblot similar to FIGS. 3 and 7. Lanes: (1) non-pretreated cells+MSA2::cA; (2) non-pretreated cells+MSA2::cD; (3) non-pretreatedcells+MSA2; (4) TCA-pretreated cells+MSA2::cA; (5) TCA-pretreatedcells+MSA2::cD; (6) TCA-pretreated cells+MSA2; and (7) non-pretreated B.subtilis (negative control). TCA pretreatment improves the binding ofMSA2::cA in a manner similar as L. lactis (i.e., compare lanes 1 and 4).Only background binding is observed for MSA2::cD and MSA2 withoutanchor.

[0050]FIG. 11. Fluorescence microscopy image of MSA2::cA binding to Lb.casei with or without TCA pretreatment. The light colored areasrepresent bound MSA2::cA. TCA pretreatment improves binding of MSA2::cAand Lb. casei.

[0051]FIG. 12. Fluorescence microscopy image of MSA2::cA and MSA2::cDbinding to M. smegmatis pretreated with TCA. The light colored areasrepresent bound MSA2 fusion protein. As illustrated, only MSA2::cAbinds.

[0052]FIG. 13. Western blot of L. lactis cells with externally boundMSA2::cA treated with LiCl or stored under different conditions. Thebands in the various lanes represent the amount of MSA2::cA thatremained bound to the TCA pretreated cells. Arrow and asterisks: as inFIG. 3. Lanes: (1) marker; (2) non-pretreated L. lactis incubatedwithout MSA2::cA; (3) non-pretreated L. lactis incubated with MSA2::cA;(4) TCA-pretreated L. lactis incubated with MSA2::cA; (5) TCA-pretreatedL. lactis incubated with MSA2::cA, subsequently washed with 8 M LiCl.;(6) TCA-pretreated L. lactis incubated with MSA2::cA, subsequentlystored in water for 3 weeks at 4° C.; (7) TCA-pretreated L. lactisincubated with MSA2::cA, subsequently stored in 10% glycerol for 3 weeksat −80° C.; and (8) TCA-pretreated L. lactis incubated with MSA2::cA,subsequently stored in water for three weeks at −80° C. As illustrated,TCA pretreatment improves binding of MSA2::cA to L. lactis cells (i.e.,compare lanes 3 and 4). Washing with 8 M LiCl and storage in water for 3weeks at 4° C. has minor effects on the bound MSA2::cA (i.e., comparelane 4 with 5 and 6). Storage at −80° C. has no effect on the boundMSA2::cA (i.e., compare lane 4 with 7 and 8).

[0053]FIG. 14. Fluorescence microscopy image of (A) MSA2::cA and (B)MSA2::cP surface expression in the recombinant strains NZ9000(pNG3041)and NZ9000(pNG3043), respectively. (C) MSA2::cA binding toTCA-pretreated L. lactis cells. The light colored areas indicate theposition of MSA2 fusion protein. The recombinant strain producingMSA2::cA has the protein on the surface in some specific spots (A). Therecombinant strain producing MSA2::cP has more on the surface organizedin several areas (B) and the surface of the TCA-pretreatednon-recombinant L. lactis with bound MSA2::cA is completely covered withthe protein (C).

[0054]FIG. 15. Western blots of L. lactis total protein extracts reactedwith rabbit immune serum diluted at 1 :100. 0: preimmune serum. 2 and 3:serum after the second and third immunization, respectively. A1:subcutaneously immunized rabbit with NZ9000ΔacmA(pNG3041) cells(recombinant, MSA2::cA surface anchored). B1: subcutaneously immunizedrabbit with NZ9000ΔacmA (negative control). C2: orally immunized rabbitwith NZ9000ΔacmA(pNG3043) cells (recombinant, MSA2::cP surfaceanchored). E1: orally immunized rabbit with TCA-pretreated NZ9000ΔacmAto which MSA2::cA had been externally bound (non-recombinant, MSA2::cAsurface anchored). The staining bands in the lanes illustrates that L.lactis proteins react with the indicated rabbit antiserum. It is visiblethat the non-recombinant TCA-pretreated strain with bound MSA2::cA (E1)evokes a minimal response to L. lactis proteins indicating that theresponse to the carrier is reduced, while the response to the malariaantigen is not negatively influenced (see, Table 2).

[0055]FIG. 16. Schematic representation of the domains in AcmA and AcmD.SS represents signal sequence. Both enzymes include a cell-wall bindingdomain that includes 3 repeats indicated by A1, 2, 3 and D1, 2, 3. Thealignments of these repeats are shown in FIG. 6. In addition, an exampleof one of the hybrid protein anchors is described in Table 5.

[0056]FIG. 17. Western blot showing the effect of pH supernatant onbinding of MSA2::cD to TCA-pretreated L. lactis cells. As previouslydescribed, the Western blot shows the amount of MSA2::cD bound by thecells. In addition, the amount of MSA2::cD that was not bound andremained in the medium after binding is shown. The arrow indicates theexpected position for pro-MSA2::cD and the asterisk indicates theposition of mature MSA2::cD. Lanes: (1) pH during binding 6.2, cells;(2) pH during binding 6.2, supernatant after binding; (3) pH duringbinding 3.2, cells; (4) pH during binding 3.2, supernatant afterbinding; and (5) positive control: L. lactis, TCA-pretreated with boundMSA2::cA at pH6.2. It is visible that MSA2::cD binds better at pH 3.2than at pH 6.2 (i.e., compare lanes 1 and 3).

[0057]FIG. 18. Western blot of medium supernatant (S) after binding toghost cells at the indicated pH's and ghost (G) with the bound proteinanchor. Lanes 1 and 2 illustrate binding at pH 3; lanes 3 and 4illustrate binding at pH 5; lanes 5 and 6 illustrate binding at pH 7.The drawing shows considerable binding at pH 5. At pH 5, the native cDanchor (D1D2D3) shows little binding. The addition of the A3 repeat,which has a high pI value, results in increased binding at pH 5.

[0058]FIG. 19. Immunization schedule. Mice immunizations were started atday 1 and repeated after 14 and 28 days. A lethal nasal challenge withS. pneumoniae was given 14 days after the last oral immunization. S.c.represents subcutaneous immunization.

[0059]FIG. 20. Serum antibody response. Mean anti-PpmA serum antibodytiters. OV represents orally immunized; IN represents intranasallyimmunized; SC represents subcutaneously immunized; Freunds PpmA refersto soluble PpmA subcutaneously administrated with Freunds completeadjuvants. High titers were obtained with the intranasally andsubcutaneously administrated Ghosts-PpmA::cA.

[0060]FIG. 21. Survival times. The orally vaccinated mice werechallenged with a lethal dose of S. pneumonia. Mice vacinnated withsoluble PpmA or Ghost alone died within 72 hours. Forty percent of themice immunized with Ghosts-PpmA::cA survived the challenge, indicatingthey were protected by the vaccination.

DETAILED DESCRIPTION EXAMPLE 1

[0061] Acid Pretreatment of Gram-Positive Bacteria Enhances Binding ofAcmA Protein Anchor Fusions.

[0062] Materials and Methods.

[0063] Bacterial Strains And Growth Conditions. Lactococcus lactisstrain MG1363 (Gasson 1983) or derivatives thereof, such as MG1363ΔacmA(Buist et al. 1995) or NZ9000ΔacmA, were used as recipients for bindingof reporter fusion protein. NZ9000 (Kuipers et al. 1997) which carriesone of the reporter plasmids was used as a production strain. L. lactisstrains were grown in M17 broth (Oxoid) supplemented with 0.5% glucosein standing cultures at 30° C. Chloramphenicol was added to the M17medium to an end-concentration of 5 μg/ml when appropriate. Forexpression, mid-log phase cultures were induced for 2 hours with theculture supernatant of the nisin producing L. lactis strain NZ9700 asdescribed by Kuipers et al. (1997). Lactobacillus casei, ATCC393, wasgrown in MRS broth (Oxoid) in standing cultures at 30° C. Mycobacteriumsmegmatis, ATCC700084, was grown in Middlebrook medium (Oxoid) at 37° C.in aerated cultures. Bacillus subtilis, 168, was grown in TY broth (perliter: 10 g tryptone, 5 g yeast extract, 5 g NaCl pH7.4) at 37° C. inaerated cultures.

[0064] Construction Of Reporter Plasmids. The merozoite surface antigen2 (MSA2) of Plasmodium falciparum strain 3D7 (Ramasamy et al. 1999)fused to the three repeats of AcmA (MSA2::cA) was used as the reporteranchor protein. The reporter anchor protein is encoded by plasmidpNG3041 based on the nisin inducible expression vector pNZ8048 (Kuiperset al. 1997) and contains a modified multiple cloning site in which thehybrid reporter gene was cloned. An in frame fusion of the reporter wasmade at the 5′ end the lactococcal PrtP signal—and prosequence, and atthe 3′ end the AcmA protein anchor sequence. The sequence of the MSA2gene that was included in the construct corresponds to nucleotides (nt)61 to 708 in Genbank accession number A06129. Primers used for theamplification of the MSA2 gene were MSA2.1(5′-ACCATGGCAAAAAATGAAAGTAAATATAGC (SEQ ID NO: 2)) and MSA2.4(5′-CGGTCTCTAGCTTATAAGCTTAGAATTCGGGATGTTGCTGCTCC ACAG (SEQ ID NO: 3)).The primers contain tags with restriction endonuclease recognition sitesthat were used for cloning. For cloning of the PrtP signal andprosequence (nt 1206 to 1766 in Kok et al. 1988), the primersPrtP.sspro.fw (5′-CCGTCTCCCATGCAAAGGAAAAAAGA AAGGGC (SEQ ID NO: 4)) andPrtP.sspro.rev (AAAAAAAGCTTGAATTCCCAT GGCAGTCGGATAATAAACTTTCGCC (SEQ IDNO: 5)) were used. The primers include restriction sites that were usedfor cloning. The AcmA protein anchor gene fragment (nt 833 to 1875) wasobtained by subcloning a PvuII-HindIII fragment from plasmid pAL01(Buist et al. 1995). Restriction endonuclease enzymes and Expand HighFidelity PCR polymerase were used in accordance with the instructions ofthe supplier (Roche). The final expression vector was designated pNG3041(FIG. 1).

[0065] A construct including a stopcodon introduced after the MSA2sequence in pNG3041 was designated pNG304. The protein secreted usingthis construct is substantially the same as the protein expressed fromthe pNG3041 plasmid except that the protein produced from pNG304 doesnot contain the AcmA protein anchor. The protein produced from pNG304 isused as a negative control in the binding assays. A vector was also madein which the AcmA protein anchor was exchanged for a protein anchor. Theputative cell-wall binding domain of L. lactis AcmD (Bolotin et al.2001) was cloned (nt 1796 to 2371 in Genbank accesssion number AE006288)using primers pACMB2 (5′-CGCAAGCTTCTGCAGAGCTCTTAGATTCTAATT GTTTGTCCTGG(SEQ ID NO: 6)) and pACMB3 (5′-CGGAATTCAAGGAGGAGAAATA TCAGGAGG (SEQ IDNO: 7)) to produce the plasmid pNG3042. pNG3042 contains an in-framefusion between MSA2 and the protein anchor of AcmD (MSA2::cD) anddiffers from plasmid pNG3041 only in the gene fragment encoding theprotein anchor.

[0066] Cell Pretreatment and Binding Conditions. Chemical pretreatmentof L. lactis NZ9000ÄacmA was done with 10% TCA (0.6 M) in the followingmanner. Cells of 0.5 ml stationary phase cultures were sedimented bycentrifugation and washed once with 2 volumes demineralized water. Cellswere resuspended in 1 volume of a 10% TCA solution and incubated byplacing the reaction tube in boiling water for 15 minutes. Subsequently,cells were washed once with 2 volumes PBS (58 mM Na₂.HPO₄.2H₂O, 17 mMNaH₂PO₄.H₂O, 68 mM NaCl; pH 7.2) and three times with 2 volumesdemineralized water. The cells were used directly for bindingexperiments or stored (as described herein) until further use.

[0067] The following chemicals and conditions were used to examine theeffect of different chemicals on the binding capacity of L. lactis cellsfor AcmA-type protein anchor fusions: acetic acid (HAc), hydrochloricacid (HCl), sulfuric acid (H₂SO₄), TCA, and trifluoroacetic acid (TFA),monochloro acetic acid (MCA). The acids were used at a finalconcentration of 0.6 M and incubated for 15 minutes in boiling water.SDS, dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO) were used ata concentration of 10%. The SDS pretreatment was incubated for 15minutes in boiling water and DMF and DMSO treatments were incubated atroom temperature for 15 minutes. Cells were also pretreated with phenol(Tris buffer saturated) and incubated for 15 minutes at 55° C. Otherchemicals pretreated at the 55° C. incubation temperature were: 4 Mguanidine hydrochloride (GnHCl), 37% formaldehyde, chloroform:methanol(CHCL₃:CH₃0H (2:1)) and 0.1% sodium, hypochlorite (NaOCl). In addition,incubation with 25 mM dithiothrietol (DTT) for 30 minutes at 37° C. anda pretreatment with hexane (100%) were analyzed.

[0068] The effect of enzymatic pretreatment of cells with lysozyme wasalso tested. For lysozyme pretreatment, the cells were resuspended inbuffer (20% sucrose, 10 mM Tris pH 8.1, 10 mM EDTA, 50 mM NaCl) withlysozyme (2 mg/ml) and incubated at 55° C. for 15 minutes. After thechemical and enzymatic pretreatments, the washing steps were the same asthe washing steps used for the TCA treated cells. TCA pretreatment ofBacillus subtilis, Lactobacillus casei and Mycobacterium smegmatis wasdone as described herein for L. lactis.

[0069] Cell-free culture supernatants containing MSA2::cA, MSA2::cD orMSA2 without anchor were incubated in four-fold excess for 10 minutes atroom temperature with pretreated cells (e.g., cells from 0.5 ml culturewere incubated with 2.0 ml culture supernatant). After binding, cellswere sedimented by centrifugation, washed twice in 2 volumesdemineralized water, resuspended in SDS-denaturation buffer, heated for5 minutes at 98° C., subjected to SDS-PAGE, and analyzed by Western blotanalysis.

[0070] Storage Conditions. Cell-free supernatants containing MSA2::cA,MSA2::cD or MSA2 were stored at −20° C. with or without 10% glycerolprior to binding. TCA pretreated L. lactis cells were stored at −80° C.in 10% glycerol prior to binding. TCA pretreated L. lactis cells withbound MSA2::cA were stored at +4° C. or −80° C. with or without 10%glycerol. Cells stored in 10% glycerol were washed once with 1 volume ofdemineralized water prior to binding.

[0071] Cell pellets (in demineralized water) of TCA pretreated L. lactiscells with or without bound MSA2::cA were frozen by contacting the vialswith liquid nitrogen and removing the water with lyophilization.Alternatively, non-frozen cell pellets were dried under vacuum at 30° C.for 2 hours prior to binding.

[0072] Western Blotting. For detection of MSA2 proteins, cell pelletscorresponding to 500 μl culture were resuspended in 50 μlSDS-denaturation buffer. Cell-free culture supernatants (1 ml) wereconcentrated by phenol-ether precipitation (Sauve et al. 1995), vacuumdried and resuspended in 50 μl SDS-denaturation buffer. Proteins wereseparated with standard SDS-PAGE techniques. After separation, proteinswere electroblotted onto PVDF membranes (Roche). In immunoblots, MSA2proteins were detected with 1:10,000 diluted rabbit MSA2-specificantiserum (Ramasamy et al. 1999) and 1:5,000 diluted anti-rabbitIgG-conjugated alkaline phosphatase (Roche) using known procedures.

[0073] Fluorescence Microscopy. 100 μl cell suspensions incubated withMSA2::cA, MSA2::cD or MSA2 fusion proteins were washed twice withdemineralized water and resuspended in an equal volume PBS containing 1%BSA and MSA2-specific rabbit antiserum diluted to 1:200. Afterincubation for 20 minutes at room temperature, the cells were washedthree times with 2 volumes PBS. Subsequently, the cells were incubatedfor 20 minutes in 1 volume PBS with 1% BSA and 1:100 diluted Oregongreen labeled goat anti-rabbit immunoglobulin G (Molecular Probes).After washing once with 2 volumes PBS and twice with 2 volumesdemineralized water, the cells were resuspended in 100 μl demineralizedwater. A 10 μl aliquot of the resuspended cells was spread onto aPolysin microslide (Menzel-Glaser), air dried, and examined under afluorescence microscope (Zeiss).

[0074] Electron microscopy. TCA-pretreated L. lactis cells incubatedwith MSA2::cA, MSA2::cD or MSA2 were collected and washed as describedherein. Immunogold labeling was performed on whole mount preparations ofglutaraldehyde fixed cells on Formvar-carbon coated nickel grids usingAuroprobe 15 nm goat anti-rabbit IgG gold marker (Amersham). Primaryantibodies against MSA2 were diluted 1:1000 in PBS-glycine buffer. Thelabeled samples were stained with 0.1% uranyl acetate (W/V in water) andexamined in a Philips CM10 transmission electron microscope at 100 kV.

[0075] Pretreatment of L. Lactis Cells With Different Chemicals. The cAprotein anchor of L. lactis AcmA can be used to bind fusion proteins toa wide variety of Gram-positive bacteria. However, the amount of fusionprotein that binds varies greatly among this group of bacteria. Bindingof MSA2::cA that covers the entire cell surface of some lactobacilli wasobserved, whereas other bacteria such as L. lactis showed only limitedlocalized binding (FIG. 2A). This phenomenon may be due to the fact thatthe cell walls of some bacterial species contain components thatinterfere with cA anchor binding. Since chemicals like SDS, TCA,chloroform/methanol and others may be used to remove components fromisolated bacterial cell walls (Morata de Ambrosini et al. 1998), theeffect of the removal of cell-wall components from L. lactis whole cellson the binding of the reporter fusion protein MSA2::cA was investigated.L. lactis cells were pretreated as described herein with variouschemicals or with lysozyme.

[0076]FIG. 3 shows typical Western blots of pretreated whole cells towhich MSA2::cA was bound. Mature MSA2::cA migrates at a position of a 75kDa protein (indicated by an asterisk). The arrow represents MSA2::cAthat contains the PrtP prosequence. The double asterisks representMSA2::cA from which one or two of the repeats have been removed. A cellmembrane anchored protease HtrA has been shown to be involved inprocessing proproteins and in removing repeats from AcmA (Poquet et al.2000). From the results of FIG. 3, it may be concluded that pretreatmentwith TCA (lanes 8 and 16 contain the same samples, the difference insignal intensity is due to differences in stain developing time), HCl,H₂SO₄ and HAc substantially improves the subsequent binding of MSA2::cA(compare with the negative control in lane 15). Other tested acids, TFAand MCA, had similar effects (not shown). Phenol, GnHCl, formamide andchloroform-/methanol pretreatments showed a moderate improvement ofbinding (lanes 4, 5, 6, 7, respectively). Minor binding improvementswere observed after pretreatment with SDS, DMF, DMSO and DTT. Theresults are summarized in Table 1. Based on the results, it appears thatpretreatment of L. lactis cells with the acids TCA, TFA, MCA, HCl, H₂SO₄and HAc are the most effective agents for improving binding of cA anchorfusion proteins to lactococcal cells. Acids such as TCA are known toremove lipoteichoic acids from cell walls.

[0077] Whether proteins are removed from the cell walls by these acidtreatments was also analyzed. FIG. 4 shows a Coomassie stained gel oflysed pretreated cells. Most of the acid treatments, except for HAc,removed a substantial amount of proteins from the lactococcal cells.Since HAc removed only trace amount of proteins (compare lane 1 and 4)and SDS pretreatment (which is known to remove proteins from the cellwalls) showed only a minor improvement of MSA2::cA binding (FIG. 3, lane1), it may be concluded that removal of proteins from the cell wall isnot critical for improving the binding of cA anchor fusions. Thisconclusion may be due to the fact that lipoteichoic acids orcarbohydrates occupy sites in the cell walls of L. lactis that interferewith efficient binding. Alternatively, acid pretreatment may result inaltering the compactness of peptidoglycan strands that make cA bindingsites more available.

[0078] TCA pretreatment was also used in all other experiments. Theoptimal TCA concentration in the boiling procedure was determined. TCApercentages of 1, 5, 10 and 20% were tested. Although 1% TCApretreatment already showed a significant improvement in binding ofMSA2::cA and 5% TCA pretreatment showed a further increase, no furtherimprovement was observed at concentrations higher than 10% TCA (FIG. 5).Therefore, the boiling procedure with 10% TCA was selected as thestandard procedure for the experiments.

[0079] The binding characteristics of the lactococcal cA homolog cD in aMSA2 fusion were analyzed using the standard TCA pretreatment procedure.Two of the three AcmD repeats are highly homologous to those of AcmA. Analignment is shown in FIG. 6. Secreted MSA2 without an anchoring domainwas included in these experiments as a negative control. In Westernblots, the effect of TCA pretreatment on the binding of MSA2::cA wasevident (FIG. 7, compare lanes 1 and 4). The effect of TCA pretreatmentwas also studied using fluorescence microscopy (FIG. 2, compare L.lactis in A and B; FIG. 8) and electron microscopy (FIG. 9, compare Aand B). Independent of the technique used, the effect of TCApretreatment on the binding of MSA2::cA can be detected.

[0080] The binding of MSA2::cD to non-TCA pretreated L. lactis cells waslow as detected in Western blots (FIG. 7, lane 2) and was undetectablein fluorescence microscopy and electron microscopy (FIG. 9A). TCApretreatment only had minor effects on the intensity of the MSA2::cDsignal in Western blots (FIG. 7, lane 5). At the same time, no MSA2::cDspecific signal associated with the pretreated cells could be observedin fluorescence microscopy (FIG. 8) and only low levels of labeling wasobserved in electron microscopy (FIG. 9C). Some cell-associated signalwas observed for MSA2 without anchoring domain for both non-TCApretreated and TCA pretreated L. lactis cells (FIG. 7, lanes 3 and 6,respectively). However, for MSA2::cD, this was not observed influorescence microscopy (not shown) and only minor labeling signals werefound in electron microscopy (FIG. 9D). Taken together, it may beconcluded that: (i) the reporter protein MSA2 does have some low degreeof affinity for bacterial cell walls that can be detected in Westernblots; (ii) the cA anchor domain specifically stimulates the binding ofthe reporter fusion to non-pretreated cells; (iii) chemicalpretreatment, especially with acids, enhances this binding; and (iv) thecD anchor domain does not promote binding of fusion proteins under theconditions applied.

[0081] The fluorescence microscopic images and electron microscopicimages of TCA pretreated lactococcal cells (FIGS. 2, 8 and 9) showedthat pretreatment leaves the integrity of the cell intact. However,cells are no longer viable (plating efficiency 0) and therefore may beconsidered as inert spherical peptidoglycan microparticles with adiameter of approximately 1 μm, “ghost cells”.

[0082] Binding to Other Gram-Positives. The binding of MSA2::cA,MSA2::cD and MSA2 without anchor domain to the Gram-positive bacteria B.subtilis, Lb. casei and M. smegmatis were also analyzed. FIG. 10 shows aWestern blot summarizing binding of MSA2::cA, MSA2::cD and MSA2 tonon-pretreated and TCA-pretreated B. subtilis cells. As for L. lactis,an increase in binding is observed for MSA2::cA. A MSA::cA specificsignal could also be visualized in fluorescence microscopy ofnon-pretreated B. subtilis cells, but with a highly improved signal forthe TCA-pretreated cells (not shown). Binding of MSA2::cD and MSA2 tonon-pretreated or TCA-pretreated cells could not be demonstrated influorescence microscopy (not shown).

[0083] Similar results were obtained for Lb. casei and M. smegmatis. Theimproved binding of MSA2::cA to TCA-pretreated Lb. casei cells is shownin FIG. 11. For MSA2::cD and MSA2, no fluorescence signals were detected(not shown). The TCA-pretreatment of M. smegmatis also had a positiveeffect on the binding of MSA2::cA, whereas no binding was observed forMSA2::cD or MSA2 (FIG. 12). Taken together, it may be concluded thatacid pretreatment, such as with TCA, improves the binding of cA proteinanchor fusions to the cell surface of Gram-positive bacteria.

[0084] Binding strength and storage conditions. The strength of theMSA2::cA binding to TCA-pretreated L. lactis cells was analyzed with atreatment of LiCl after the binding. LiCl is commonly used to removeproteins from bacterial cell walls. From the Western blot of FIG. 13, itmay be concluded that 8 M LiCl partially removes MSA2::cA from the L.lactis cells (compare lanes 4 and 5). Therefore, although MSA2::cA bindsnon-covalently to cell walls, the binding interactions are most likelyvery strong.

[0085] Cell-free culture supernatants with MSA2::cA were stored with orwithout 10% glycerol at −20° C. MSA2::cA stored in this manner forseveral weeks had the same capacity to bind to TCA-pretreated L. lactiscells (not shown).

[0086] TCA-pretreated L. lactis cells with bound MSA2::cA were storedfor 3 weeks at +4° C. in demineralized water or at −80° C. indemineralized water with or without 10% glycerol. The samples wereanalyzed in Western blots. Storing pretreated cells with bound MSA2::cAfor 3 weeks in water at +4° C. only resulted in a loss of signal ofabout 50% (FIG. 13, compare lanes 4 and 6). Whether this loss of signalwas due to degradation or due to release of the protein into the waterwas not determined. Storage at −80° C. with or without 10% glycerol hadno effect on the binding (FIG. 13, compare lanes 4, 7 and 8).

[0087] In addition, the effects of drying and lyophilization on thebinding of MSA2::cA to TCA-pretreated L. lactis cells were studied.Drying of pretreated cells had no observable negative effect on bindingof MSA2::cA afterwards. Dried pretreated cells with bound MSA2::cA couldbe resuspended in water without losing bound fusion protein. This wasalso observed for lyophilized cells with bound MSA2::cA. Lyophilizationof TCA-pretreated cells prior to binding resulted in loss of the bindingcapacity for MSA2::cA (results not shown).

[0088] From these data, it may be concluded that: (i) in spite of thenon-covalent character of cA anchor binding to cell walls, the bindingis very strong; (ii) cell-free culture supernatants can be stored safelyat −20° C.; and (iii) drying of TCA-pretreated cells provides anefficient and simple method for storage of such cells either with orwithout bound cA-anchor fusions.

EXAMPLE 2

[0089] Oral Immunizations of Rabbits with Non-Recombinant Lactococcuslactis Preloaded with the Plasmodium falciparum Malaria Antigen MSA2Fused to the Lactococcal AcmA Protein Anchor.

[0090] In Example 1, a technology is described that efficiently bindsprotein hybrids when externally added to the cell surface ofnon-recombinant gram-positive bacteria by means of an AcmA-type proteinanchor. This technology provides the possibility to provide bacteria orbacterial cell walls with new traits without introducing recombinant DNAinto them. The immunogenicity in rabbits of the Plasmodium falciparummerozoite surface protein, MSA2 of strain 3D7 (Ramasamy et al. 1999),presented on the cell surface of non-recombinant non-living L. lactiscells as an AcmA anchor fusion protein was investigated.

[0091] Materials and Methods.

[0092] Bacterial Strains and Growth Conditions. The L. lactis strainwhich produces MSA2::cA, the strain's growth conditions, the inductionfor expression, the TCA pretreatment of the L. lactis recipient cellsand the binding of MSA2::cA to the cells was described in example 1 withthe following modification: a ratio of 1 (TCA-pretreated cells) to 5(cell-free culture supernatant with MSA2::cA) was used for binding. AnL. lactis NZ9000 strain carrying plasmid pNG3043 was used as a positivecontrol in the immunization experiments (was positive in a previousunpublished experiment). Plasmid pNG3043 encodes an MSA2 hybrid proteinthat contains the lactococcal PrtP cell-wall anchoring domain at itsC-terminus (MSA2::cP) instead of the AcmA protein anchor. The PrtPcell-wall anchoring domain contains the LPXTG (SEQ ID NO: 1) motif thatenables a membrane-linked sortase to covalently couple the protein tothe cell wall (Navarre and Schneewind 1994). The cP domain used inconstruct pNG3043 corresponds to nt 6539 to 6914 in Kok et al. (1988).Primers used for the amplification of this fragment were PrtP.cwa.fw3(5′-ATATAAAGCTTGCAAAGTCTGAAAACGAAGG (SEQ ID NO: 8)) and PrtP.cwa.rev(5′-CCGTCTCAAGCTCACTATTCTTCACGTTGTTTCCG (SEQ ID NO: 9)). The primersinclude restriction endonuclease recognition sites for cloning. PlasmidpNG3043 differs from plasmid pNG3041 in the cell-wall binding domain.Growth conditions and induction of expression of strainNZ9000ΔacmA(pNG3043) were the same as for strain NZ9000 ΔacmA(pNG3041).

[0093] Rabbit Immunizations. Ten barrier-reared, New Zealand whiterabbits obtained from Harlan laboratories, The Netherlands, were used ingroups of 2 for experimental immunizations. The care and use of animalswere according to WHO guidelines (WHO/LAB/88.1). The rabbits were earbled prior to immunization to obtain preimmune sera. Details of therabbits and immunogens are as follows:

[0094] Rabbits A1 and A2 were subcutaneously immunized withNZ9000ΔacmA(pNG3041) cells (recombinant, MSA2::cA partly surfaceanchored).

[0095] Rabbits B1 and B2 were subcutaneously immunized with NZ9000ΔacmA(negative control).

[0096] Rabbits C1 and C2 were orally immunized with NZ9000ΔacmA(pNG3043)cells (recombinant, MSA2::cP surface anchored).

[0097] Rabbits D1 and D2 were orally immunized with NZ9000ΔacmA(pNG3041)cells (recombinant, MSA2::cA surface anchored).

[0098] Rabbits E1 and E2 were orally immunized with TCA treatedNZ9000ΔacmA to which MSA2::cA had been bound from NZ9000ΔacmA(pNG3041)culture supernatant (non-recombinant, MSA2::cA surface anchored).

[0099] Stocks of NZ9000ΔacmA(pNG3043) with MSA2::cP expressed at itssurface were stored in aliquots of 10¹¹ cells in growth mediumcontaining 10% glycerol at −80° C. The cells remain viable under theseconditions and retain MSA2 on the surface as demonstrated byimmunofluorescence (not shown). The first immunization was carried outwith freshly grown bacteria. For subsequent immunizations, stocks ofbacteria were freshly thawed, washed and resuspended in buffer at theappropriate concentration for immunizations.

[0100] On the other hand, the non-pretreated NZ9000ΔacmA (negativecontrol), the non-pretreated NZ9000ΔacmA(pNG3041) and the TCA-pretreatedNZ9000ΔacmA with the externally bound MSA2::cA were prepared daily fromfresh cultures.

[0101] Subcutaneous injections were performed with a total of 5×10⁹cells in 100 μl PBS without any adjuvant into two sides on either sideof the spine. The subcutaneous injections were repeated two more timesat 3 week intervals. Prior to oral immunization, the rabbits weredeprived of water and food for 2-4 hours. The rabbits were then fed5×10¹⁰ cells resuspended in 1 ml of 0.5% sucrose. Each dose was repeatedfor three successive days to obtain reproducible oral immunization.Altogether, three series of oral immunizations were given at 3 weekintervals. Adverse effects consequent to the immunizations, includinggranulomas at the sites of subcutaneous injections, were not observedindicating that L. lactis was well tolerated by the animals.

[0102] Serum Antibody Responses. Rabbits were ear bled 2 weeks aftereach immunization to obtain sera for antibody assays. The sera werestored at −20° C. until use. Ten-fold serial dilutions of the antiserain 2% BSA in PBS were used in immunofluorescence assays (IFA) todetermine the titer of the antibodies against MSA2 on the surface of 3D7P. falciparum merozoites. IFA was performed on acetone-methanol fixedlate stage 3D7 P. falciparum parasites as previously described (Ramasamy1987). For detection of antibody isotypes, Oregon Green conjugated goatanti-rabbit Ig (Molecular Probes) was used as the second antibody. Fordetection of IgG antibodies, a fluorescein conjugated, affinitypurified, mouse monoclonal with specificity against rabbit IgG chains(Rockland) was used.

[0103] Results and Discussion.

[0104] Surface Expression of MSA2 in Different L. Lactis Strains.Coomassie staining of SDS-PAGE gels and fluorescence microscopy wereused to determine, in a semi-quantitative way, the number of MSA2molecules expressed and surface exposed by the recombinant lactococcalstrains carrying plasmid pNG3041 or pNG3043 that produce MSA2::cA orMSA2::cP, respectively, and by the non-recombinant TCA-pretreated L.lactis cells to which MSA2::cA had been bound from the outside. Therecombinant strains were estimated to produce approximately 1.4×10⁵molecules of MSA2::cA or MSA2::cP. The surface exposure of MSA2::cA andMSA2::cP differed considerably as shown by fluorescence microscopy inFIG. 14. The non-recombinant TCA-pretreated L. lactis cells with boundMSA2::cA showed a uniform staining of the entire cell surface. However,the semi-quantitative SDS-PAGE analysis indicated that about 1×10⁴molecules of MSA2::cA per cell were represented.

[0105] Accordingly, it may be concluded that the number of surfaceexposed MSA2::cA and MSA2::cP on the recombinant lactococcal strains isless than 10% of the total number of molecules produced by thesestrains. The other molecules are most likely trapped in the membrane orthe cell wall. Similar observations were made by Norton et al. (1996)for the expression of TTFC fused to the cP cell-wall anchoring domain.In that study, only membrane-associated or cell-wall-associated TTFCcould be demonstrated and no surface-exposed TTFC::cP was demonstrated.Thus, it appears that binding from the outside to TCA-pretreated cellsis a more efficient method to surface-expose proteins on L. lactiscells.

[0106] Anti-MSA2 Antibody Responses In Orally Immunized Rabbits.Characteristics of the anti-MSA2 antibody response to the immunizationsare summarized in Table 2. The oral immunizations with the recombinantL. lactis that produces MSA2::cP (rabbits C1 and C2) were done before(unpublished results) and used as a positive control. In the previousexperiment, a similar antibody response was found. The presentexperiment showed that specific antibodies against near native MSA2 weredetectable after two immunizations for group A, D and E rabbits, andthat antibody titers increased in all instances after a thirdimmunization. IgG antibodies were predominant after three immunizationsin either the subcutaneous or oral route. A comparatively weak anti-MSA2surface IFA, attributable to the generation of cross-reactive antibodies(as described herein), was also observed after three controlsubcutaneous immunizations with L. lactis cells alone.

[0107] Taken together, the results indicate that: (i) MSA2 produced bylactococcal cells elicits serum antibodies that recognize native P.falciparum parasite MSA2; (ii) MSA2-specific T_(h) cells are activatedthrough mucosal immunization due to the presence of systemic IgGantibodies (Table 2) that can be boosted (unpublished results); and(iii) oral immunizations with MSA2::cA bound to non-recombinantnon-living TCA-pretreated L. lactis cells are as efficient in evokingspecific serum antibody responses as the live recombinant strainproducing MSA2::cA that was administered subcutaneously or orally, or asefficient as the live recombinant strain producing MSA2::cP that bindsMSA2 covalently to its cell wall delivered orally.

[0108] Anti-Lactococcal Antibody Responses. Western blots (FIG. 15)demonstrated significant antibody responses against L. lactis antigensafter two and three immunizations of the rabbits. The responses werenotably greater after subcutaneous (group A and B rabbits) than oralimmunization with L. lactis (group. C rabbits). Oral immunization withthe TCA-pretreated lactococcal cells (group E rabbits) elicitedantibodies that reacted at a lower intensity with fewer L. lactisantigens than oral immunization with viable L. lactis cells. This ismost likely due to the fact that proteins are removed from thelactococcal cells by the TCA pretreatment (see, example 1). The loweranti-carrier response observed for the TCA-pretreated (non-recombinant)cells renders this type of delivery vehicle more suitable for repeatedimmunization strategies than its untreated (recombinant) counterpart.

EXAMPLE 3

[0109] pH-Dependent Cell-Wall Binding of AcmA Protein Anchor Homologsand Hybrids.

[0110] The cell-wall binding domain or anchor of the lactococcalcell-wall hydrolase AcmA includes three repeats of 45 amino acids thatshow a high degree of homology (Buist et al. 1995). These three repeatsbelong to a family of domains that meet the consensus criteria asdefined in PCT publication WO 99/25836 and can be found in varioussurface located proteins in a wide variety of organisms. Another featurethat most of these domains have in common is that their calculated pIvalues are high, approximately 8 or higher (Table 3). The pH used inprevious binding experiments with MSA2::cA (i.e., examples 1 and 2) wasapproximately 6, indicating that the binding domain was positivelycharged.

[0111] The AcmA protein anchor homolog of the lactococcal cell-wallhydrolase AcmD (cD) (Bolotin et al. 2001) also includes three repeats(FIG. 16) with a calculated pI that is lower (approximately pI 3.8) thanthat of the cA domain (Table 4). Consequently, the cD anchor wasnegatively charged at the binding conditions used in example 1. Nobinding of the MSA2::cD reporter protein occurred under these conditionsas demonstrated herein. Therefore, the influence of the pH duringbinding of a cD fusion protein (MSA2::cD) was investigated. Furthermore,a hybrid protein anchor including the three cD repeats and one cA repeatthat has a calculated pI value that is higher than that of the cDrepeats alone was constructed. The hybrid protein anchor showed betterbinding pH values above the pI of the cD repeats alone, indicating thatthe pH binding range of AcmA-type protein anchors can be manipulated byusing the pI values of the individual repeats in hybrids.

[0112] Materials and Methods.

[0113] Bacterial strains, growth and induction conditions, TCApretreatment of L. lactis cells, incubation of the MSA2 protein anchorfusion proteins to TCA-pretreated cells, washing conditions, protein gelelectrophoresis, Western blotting and immunodetection were the same asdescribed herein with reference to example 1. The cell-free culturesupernatants with MSA2::cA, MSA2::cD or A3D1D2D3 have a pH ofapproximately 6.2. The influence of pH was examined by adjusting the pHof the cultures by the addition of HCl or NaOH to obtain the requiredpH.

[0114] Plasmid Constructions. The plasmid that expresses the MSA2::cDfusion was described herein with reference to example 1. Plasmid pPA43is based on the same expression plasmid and contains an in frame fusionof the lactococcal signal sequence of Usp45 (ssUsp; van Asseldonk et al.1990. Gene 95: 155-160), the c-myc epitope for detection purposes, theA3 cA repeat and repeats D1, D2 and D3 of cD. Primers used for cloningA3 were cArepeat3.fw (CCG TCT CCA ATT CAA TCT GCT GCT GCT TCA AAT CC(SEQ ID NO: 10)) and cA repeat3.rev (TAA TAA GCT TAA AGG TCT CCA ATT CCTTTT ATT CGT AGA TAC TGA CCA ATT AAA ATA G (SEQ ID NO: 11)) (the primersinclude the A3 specific sequences). The primers used for cloning thethree cD repeats were cDrepeat1.fw(CCGTCTCCAATTTCAGGAGGAACTGCTGTTACAACTAG) (SEQ ID NO: 12) andcDrepeat3.rev (TAATAAGCTTAAAGGTCTCCAATTCCAGCAACTTGCAAAACTTCTCCT AC) (SEQID NO: 13) (the primers include the cD specific sequences).

[0115] Results and Discussion.

[0116] Binding of MSA2::cD at Low pH. Since binding of MSA2::cD was notobserved at a pH (the pH of the culture medium after- growth andinduction is about 6.2) higher than the calculated pI for the cD domain(i.e., pI 3.85), binding was studied when the pH of the medium wasadjusted to pH 3.2. TCA-pretreated L. lactis cells were used as thebinding substrate and the relative amounts of bound MSA2::cD wereanalyzed in Western blots. The amounts of unbound reporter proteinremaining in the culture supernatant after binding were also analyzed.FIG. 17 shows a clear increase in bound MSA2::cD when binding isperformed at pH 3.2 (compare lanes 1 and 3). At the same time, lessunbound reporter protein remained in the supernatant (compare lanes 2and 4). This result indicates that positive charges are important forbinding of cA-type anchoring domains.

[0117] Binding of cAcD Hybrid Anchors. Analysis of the pI values of thecA homologs in Table 3 indicates that two classes of repeats can bedistinguished: a majority (99 out of 148) of homologs that have a highpI value (>8) and a smaller group (33 out 148), of which cD is arepresentative, that has pI values lower than 6. Based on theexperimental results, it is shown that these types of anchoring domainsonly bind to bacterial cell walls at a pH that is lower than theanchoring domains pI. Notably, most cell-wall binding domain homologsinclude repeats with a pI that are representatives of one of the twogroups, i.e., only repeats with a high or low pI. Some proteins withcell-wall binding domains, e.g., those of DniR of Trepanoma pallidum andan amidase of Borrelia burgdorferi, include repeats with high and lowpI. Since the binding pH of such “natural hybrid” cell-wall bindingdomains is below the intermediate pI value of the total number ofrepeats present in the domain, a hybrid cell-wall protein anchor wasconstructed using the cA and cD repeats with an intermediate pI value.Table 5 lists the native AcmA and AcmD anchors and a number of examplesof cA/cD hybrids. The constructed hybrid protein anchor (A3 D1D2D3) hasa calculated pI value of approximately 5.1. A protein anchor includingonly D1D2D3 shows little binding at a pH above its calculated pI (asdescribed herein). The A3 (pI 10) domain shows similar binding at pH 5and pH 7.

[0118] The binding of the hybrid anchor A3D1D2D3 was tested at pH 3, pH5 and pH 7. At pH 3, most protein had been bound to the ghost cells(FIG. 18). At pH 5, there was considerable binding (±40%), whereas therewas only minimal binding at pH 7 (±20%). This result indicates the pHrange of binding for eD repeats was shifted to higher pH values by theaddition of one cA repeat (A3) that caused a shift in calculated pIvalues of 3.8 to 5.1. The increase of binding at pH 5 for the A3D1D2D3hybrid cannot be attributed to binding of the A3 repeat alone. If thiswas the case, then the same level of binding should occur at pH 7 sincethe A repeats show the same binding at these pH values. In addition, theincreased binding at pH 5 is not an additive effect in the sense that anextra binding domain results in increased binding. It has previouslybeen shown that addition of one repeat to the cA anchor did not resultin increased binding. The binding at the higher pH values of theA3D1D2D3 repeats as compared to the D1D2D3 repeats alone, thus may beattributed to the increase in the calculated pI value of the hybridcA/cD anchor. This demonstrates that pH binding properties of thesetypes of protein anchors may be manipulated on the basis of the pIvalues of individual repeats present in the hybrid anchor.

EXAMPLE 4

[0119] Induction of Cellular Immune Responses in Mice after OralImmunizations with Lactococcal Ghosts Displaying the Malaria Plasmodiumfalciparum Antigen MSA2 Fused to the Lactococcal AcmA Protein Anchor.

[0120] Non-genetically modified non-living Lactococcus lactis cells(ghosts) preloaded with the Plasmodium falciparum MSA2 antigen fused tothe AcmA protein anchor (MSA2::cA) were used to orally immunize mice ina similar way as described herein with reference to example 2. In thisexperiment, the question of whether immunizations through the oral routewith the non-recombinant non-living Ghosts carrying MSA2::cA on theirsurface (Ghosts-MSA2::cA) can elicit typical Th1-type immune responses,such as IgG2 antibodies and gamma-interferon (γIFN) producing T cells inthe spleen is addressed. These responses are particularly relevant toobtain immunity for pathogens, such as malaria, that undergo stages intheir life cycle where they are not in the blood, but hide in cells.

[0121] Materials and Methods.

[0122] Groups of five mice of different strains were used forimmunization. The strains of mice used were Balb/c (with the majorhistocompatibility locus allotype of H2d), C57 Black (H2b), C3H (H2k)and ICR (out bred, i.e., of varying H2 types). Oral immunizations wereperformed at three weekly intervals. Immunizations were performed withMSA2::cA absorbed on to the surfaces of TCA treated Lactococcus lactiscells (Ghosts-MSA2::cA) or with recombinant L. Iactis that displayedMSA2 on the surface through the use of a covalently linked cell-wallanchor (L. lactis(MSA2::cP)) as described herein with reference toexample 2. The mice were tail bled to obtain serum samples two weeksafter the second, third and fourth immunizations. Fecal pellets werecollected and extracted to examine intestinal IgA antibody production.The mice were sacrificed at the end of each experiment and the spleenswere removed for examining T-cell responses by ELISPOT. MSA2-his tagproduced in E. coli was used as antigen in the ELISA and ELISPOT assay.The growth of bacterial strains and the preparation of Ghost cells wasas described herein with reference to example 2.

[0123] Results and Discussion

[0124] Kinetics and Isotypes of the Serum IgG Antibodies Generated OralImmunizations. Differences in the kinetics of the antibody response andthe isotype distribution were observed between different murine strains.The antibody response was also different when living recombinant L.lactis (MSA2::cP) or Ghosts-MSA2::cA were used as immunogens. WithGhosts-MSA2::cA, high serum antibody levels were detectable in the C3Hmice after two immunizations. IgG antibodies were detectable in all fourmurine strains after three and four immunizations. Antibody titers werehighest in C3H mice. IgG antibodies that reacted with native MSA2 onparasites were detected in the sera of immune mice by fluorescencemicroscopy (IFA) confirming that the immunizing form of the proteinelicits biologically relevant antibodies. Control immunizations wereperformed with Ghosts alone where no MSA2-specific antibodies wereelicited. In parallel experiments using MSA2cP as the immunogen, highserum IgG antibody levels were only seen with Balb/c mice after twoimmunizations. After three and four immunizations, good antibodyresponses developed in C3H mice. Antibody titers were highest in Balb/cmice.

[0125] Significant differences existed between the strains in theisotypes of the elicited serum IgG antibodies in response toimmunization with Ghosts-MSA2::cA. Balb/c mice showed higher levels ofIgG2a and IgG2b antibodies, some IgG3 antibodies and negligible IgG1which demonstrates a possible Th1 bias. On the other hand, C57 Black andC3H mice had high IgG1, IgG2a and IgG2b, and lower IgG3 antibodies toMSA2 which is more characteristic of a mixed Th1 and Th2 response. ICRmice, as expected, showed a range of responses. Some ICR mice had theBalb/c and others the C3H/C57 Black pattern of IgG isotypes.

[0126] Formation of Mucosal Antibodies. IgA antibodies were detected byELISA in the fecal pellets of the ICR and Balb/c mice, but were notdetected in C3H or C57 Black mice when immunization was performed withliving recombinant L. lactis(MSA2::cP) or Ghost-MSA2::cA.

[0127] T-Cell Responses. The increase of the intensity of the IgG ELISAreactions seen in mice immunized with Ghosts-MSA2::cA with eachimmunization demonstrates that boosting takes place and that aTh-dependent antibody response exists in these animals. The IgG isotypedistribution further confirms this conclusion. Therefore, Th cells aregenerated in ICR, Balb/c, C57 Black and C3H mice.

[0128] The ELISPOT assay for detecting gamma-interferon (γIFN) producingcells detects mainly CD8⁺ Tc cells, which are an important component ofthe immune response to many pathogens, including malaria parasites.His-tagged MSA2 produced in E. coli was used as antigen in the assay.MSA2-specific γIFN producing cells could be detected in the spleens ofBalb/c, C57 Black and C3H mice that were immunized with Ghosts-MSA2::cA.MSA2-specific γIFN producing cells were not observed in the spleens ofcontrol mice immunized with Ghosts alone or with the living recombinantL. lactis(MSA2-cP). The latter group showed a high level of non-specificγIFN producing cells. The high background observed may be due to ongoinginflammation.

[0129] The sensitization of MSA2-specific Tc cells in the spleen afterimmunization with the non-recombinant non-living L. lactis Ghost-systemcarrying a foreign protein is a novel finding which is applicable tomalaria since protection against sporozoite-infection is associated withγIFN producing cells being produced in the spleen.

[0130] The non-recombinant non-living Ghost system can be used in oralimmunizations to elicit typical Th1-type immune responses. These typesof responses are particularly relevant to obtain immunity for pathogensthat undergo stages in their life cycle where the pathogens are not inthe blood, but rather hide in cells. The responses are more pronouncedand more specific for the Ghost system than for the living recombinantsystem. The Ghost system has the additional advantage of eliminating therisk of spreading recombinant DNA into the environment.

EXAMPLE 5

[0131] Protection of Mice for Lethal Streptococcus pneumoniae Challengeafter Oral Immunizations with Lactococcal Ghosts Preloaded with PpmAAntigen Fused to the Lactococcal AcmA Protein Anchor.

[0132]Streptococcus pneumoniae is the leading etiological agent ofsevere infections including septicemia, meningitis, pneumonia, andotitis media. Recent studies on the molecular epidemiology andpathogenesis of S. pneumoniae have identified pneumococcal proteins withvaccine potential. One of these proteins, the protease maturationprotein PpmA, has been shown to elicit immune protective potential in amouse pneumonia model.

[0133] The non-genetically modified lactococcal ghosts have been shownto be an efficient carrier for use in oral immunizations of rabbits andmice in order to elicit strong anti-malaria immune responses. Theconstruction of lactococcal ghosts that display the S. pneumoniae PpmAfused to the lactococcal AcmA cell-wall binding domain on their surfaceis described herein. The ability of these ghosts to protect orallyimmunized mice from a lethal nasal dose of S. pneumoniae wasinvestigated.

[0134] Materials and Methods.

[0135] Bacterial Strains and Growth Conditions. L. lactis was grown andghost cells were prepared as described herein with reference toexample 1. S. pneumoniae was grown as described before (Gingles et al.2001. Infect Immun 69: 426-434).

[0136] Construction ppmA Protein Anchor Fusion Expression Plasmid. Theexpression plasmid for ppmA protein anchor fusion (PpmA::cA) wassubstantially similar to the expression plasmid for the MSA2 proteinanchor fusion as described herein with reference to example 2. For thesecretion of PpmA::cA, the secretion signal sequence of the Usp45protein (ssUsp) of L. lactis (van Asseldonk et al. 1990. Gene 95:155-160) was used. The PpmA gene was cloned by PCR using primers ppmA. 1(CGGTCTCACATGTCGAAAGGGTCAGAAGGTG CAGACC) (SEQ ID NO: 14) and ppmA.2(CGGTCTCGAATTGCTTCGTTTGATGTACTACTG CTTGAG) (SEQ ID NO: 15) resulting inplasmid pPA32 which contains ppmA as an in frame fusion with ssUsp45 andthe protein anchor (ssUsp::ppmA::cA). Expression of the fusion generesults in the secreted product PpmA::cA. The primers include an Eco31Irestriction enzyme recognition site that was used for digestion of thePCR fragment. This restriction digest produced NcoI and EcoRI stickyends which were used for cloning. The primers also iuncluded the ppmAsequences. Chromosomal DNA of S. pneumoniae strain D39 was used as atemplate for the PCR reactions.

[0137] Preparation of the Vaccine. Three liters of M17 medium withPpmA::cA, obtained after growth and used to induce producer cells forexpression of L. lactis (pPA32), was centrifuged and filter sterilized(0.2 μm) to remove the producer cells. Ghost cells were prepared from0.5 liter of L. lactis NZ9000(ΔacmA). After binding, the ghost cellswith PpmA::cA (Ghosts-PpmA::cA) were isolated by centrifugation andwashed with PBS. The ghost cells were stored in PBS in aliquots of2.5×10¹⁰ Ghosts/ml at ‘80° C. Two control groups included: (i) Ghostswithout bound PpmA::cA; for the sample preparation the same amounts ofghost cells were used and the same centrifugation and washing steps wereperformed, but the binding step was omitted; and (ii) soluble PpmA wasisolated as a his-tagged fusion.

[0138] Mice Immunizations. Groups of 10 mice (CD-1) were used in theimmunizations. Oral doses included 5×10⁹ Ghosts with or without PpmA::cA(50 μg) or 50 μg soluble PpmA in PBS. Nasal doses included 5×10⁸ Ghostswith or without PpmA::cA (5 μg) or 5 μg soluble PpmA. 10⁸Ghosts-PpmA::cA (1 μg) were subcutaneously injected. For intranasalimmunizations, the mice were slightly anesthetized with Isofluorane.

[0139] Intranasal Challenge. The groups of orally immunized mice wereintranasally challenged 14 days after the last booster immunization witha dose of 10⁶ colony forming units (CFU) S. pneumoniae D39 as described(Kadioglu et al. 2000 Infect Immun 68: 492-501). Mice were monitoredafter the challenge for visible clinical symptoms for 7 days, at whichpoint the experiment was ended. Mice that were alive after 7 days wereconsidered to have survived the pneumococcal challenge and mice thatbecame moribund during the 7-day period were judged to have reached theendpoint of the assay. The time the animal became moribund was recorded,and the animal was sacrificed by cervical dislocation.

[0140] ELISA Analysis. Serum samples were taken from each mouse beforethe intranasal challenge and stored at −20° C. before use. Microtiterplates were coated with 100 μg PpmA/ml in 0.05 carbonate buffer. Serial10-fold dilutions of pooled serum of each group were incubated on theplates as described (Gingles et al. 2001, Infect. Immun. 69: 426-434).Anti-mouse immunoglobulin-horse-radish peroxidase conjugate was used fordetection and the absorbance was measured at 492 nm.

[0141] Results and Discussion.

[0142] Serum Antibody Response. Mice were immunized orally, nasally andsubcutaneously according to the scheme shown in FIG. 19. Anti-PpmAantibody titers in the blood serum were determined for each group byELISA assays. The results are given in FIG. 20. As expected, ghostsalone administered orally or nasally, OV Ghosts or IN Ghosts,respectively, did not induce anti-PpmA antibodies. Soluble PpmA given bythe nasal route resulted in only a low anti-PpmA antibody titer whichagrees with the general findings that soluble antigens are not veryimmunogenic when given by the mucosal routes. Ghosts-PpmA::cA providedby the oral route (OV PpmA+Ghost) induced only a low level of anti-PpmAserum antibodies. This contrasts the results for the oral immunizationexperiments described herein with reference to examples 2 and 4 withMSA2::cA. However, the contrast may be antigen-type related.

[0143] Intranasal administration of Ghosts-PpmA::cA resulted in a hightiter of anti-PpmA antibodies (IN PpmA+Ghosts). A high titer was alsoobtained by subcutaneous administration of Ghosts-PpmA::cA. These titerswere lower by a factor of 5 to 10 when compared to soluble PpmA that wassubcutaneously administered and formulated with the strong Freundscomplete adjuvant (Peter Adrian, Erasmus University Rotterdam, TheNetherlands, unpublished results). In addition, the Freunds PpmA vaccinecontained 50 μg PpmA per dose, whereas the intranasally administeredGhosts-PpmA:cA contains only 5 μg/dose and the subcutaneous Ghost-PpmA::cA vaccine contains only 1 μg PpmA/dose. This result demonstrates theadjuvant effect of the ghost cells. Side effects of the orally, nasallyor subcutaneously administrated ghosts were not observed, which is incontrast to the severe side effects that are usually seen with the useof Freunds adjuvants.

[0144] The results demonstrate that high titer serum antibodies can beobtained by the mucosal route of adminstration. These data also showthat ghost cells may be safely used in traditionally injected vaccineswithout side effects in order to induce high titer serum antibodies.

[0145] Protection Against Challenge. The mice orally immunized withsoluble PpmA, Ghosts alone or Ghosts-PpmA::cA were challenged 14 dayspost immunization with a lethal intranasal dose of S. pneumoniae. Themice immunized with soluble PpmA or Ghosts alone died within 72 hoursafter challenge. The group immunized with Ghosts-PpmA::cA showed asurvival rate of 40% (FIG. 21). This results shows that mucosalimmunization of mice with Ghosts-PpmA induces protective immunityagainst a lethal S. pneumoniae challenge. In conclusion, thenon-recombinant non-living Ghost system may be used to elicit high titerserum antibodies and the mucosal route of administration may be used toobtain protective immunity against a mucosally acquired pathogen.

[0146] References:

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[0153] Navarre and Schneewind (1994) Mol. Microbiol. 14: 115-121.

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[0161] TABLE 1 Effect of different pretreatments of L. lactis on bindingof MSA2::cA. Treatment Signal on Western blot H₂O − 10% TCA (0.6 M) ++++0.6 M HAc ++++ 0.6 M HCl ++++ 0.6 M H₂SO₄ ++++ 0.6 M TFA ++++ 0.6 M MCA++++ Phenol ++ 4 M GnHCl ++ 37% formaldehyde ++ CHCl₃/MeOH ++ 10% SDS +10% DMF + 10% DMSO + 25 mM DTT + 0.1% NaHClO* − Hexane − Lysozyme* −

[0162] TABLE 2 MSA2 antibody titres of rabbit serum determined by IFA onPlasmodium falciparum 3D7 asexual blood stage parasites. ImmunisationRabbit 3^(rd) serum Immunogen P 2^(nd) IgG A1 s.c. L. lactis[MSA2::cA] 02 4 4 A2 s.c. L. lactis[MSA2::cA] 0 2 5 5 B1 s.c. L. lactis 0 0 1 n.d.B2 s.c. L. lactis 0 0 1 n.d. C1 oral L. lactis[MSA2::cP] 0 3 5 5 C2 oralL. lactis[MSA2::cP] 0 2 5 5 D1 oral L. lactis[MSA2::cA] 0 2 4 5 D2 oralL. lactis[MSA2::cA] 0 2 4 5 E1 oral TCAL. lactis + MSA2::cA 0 2 5 5 E2oral TCAL. lactis + MSA2::cA 0 2 5 4

[0163] TABLE 3 AcmA cell wall binding domain homologs and theircalculated pI values. (the pI values are indicated directly behind theamino acid sequences) Lactococcus *acmA YTVKSGDTLWGISQRYG SEQ ID NO:169.75 245-287(33) 437 U1769600 muramidase lactis ISVAQIQSANNLKST IIYIGQKLVLT VKVKSGDTLWALSVKYK SEQ ID NO:17 9.64 321-363(31)TSIAQLKSWNHLSSD T IYIGQNLIVS HKVVKGDTLWGLSQKSG SEQ ID NO:18 10.06395-437 SPIASIKAWNHLSSD T ILIGQYLRIK *acmD YKVQEGDSLSAIAAQYG SEQ IDNO:19 4.15 194-237 QGCI25 TTVDALVSANSLENAND IHVGEVLQVA YTVKSGDSLYSIAEQYGSEQ ID NO:20 3.78 258-303 MTVSSLMSANGIYDVNS MLQVGQVLQVTVYTIQNGDSIYSIATANG SEQ ID NO:21 4.15 319-361 MTADLAALNGFGIND M IHPGQTIRITuc2009 *lys YVVKQGDTLSGIASNWG SEQ ID NO:22 6.31 332-375(10) 428 L31364glycosidase TNWQELARQNSLSNPNM (muramidase) IYAGQVISFT YTVQSGDNLSSIAILLGSEQ ID NO:23 3.45 386-428 TTVQSLVSMNGISNPNL IYAGQTLNY -LC3 *lysBYIVKQGDTLSGIASNLG SEQ ID NO:24 6.31 333-376(10) 429 U04309 muramidaseTNWQELARQNSLSNPNM IYSGQVISLT YTVQSGDNLSSIARRLG SEQ ID NO:25 8.79 387-429GISNPNLIYAGQTLNY Enterococcus *autolysin YTVKSGDTLNKIAAQYG SEQ ID NO:269.74 363-405(25) 671 P37710 muramidase faecalis VSVANLRSWNGISGD LIFVGQKLIVK YTVKSGDTLNKIAAQYG SEQ ID NO:27 9.74 431-473(25)VTVANLRSWNGISGD L IFVGQKLIVK YTIKSGDTLNKIAAQYG SEQ ID NO:28 9.74499-541(25) VSVANLRSWNGISGD L IFAGQKIIVK YTIKSGDTLNKISAQFG SEQ ID NO:299.85 567-609(19) VSVANLRSWNGIKGD L IFAGQTIIVK HTVKSGDSLWGLSMQYG SEQ IDNO:30 9.35 629-671 ISIQKIKQLNGLSGD T IYIGQTLKVG hirae *mur2YTVKSGDSVWGISHSFG SEQ ID NO:31 9.35 257-299(38) 666 P39046 muramidaseITMAQLIEWNNIKNN F IYPGQKLTIK YTVKSGDSVWKIANDHG SEQ ID NO:32 7.14338-380(33) ISMNQLIEWNNIKNK F VYPGQQLVVS YTVKAGESVWSVSNKFG SEQ ID NO:339.91 414-456(32) ISMNQLIQWNNIKNN F IYPGQKLIVK YTVKAGESVWGVANGIS SEQ IDNO:34 9.64 489-531(33) MNQLIEWNNIKNN FIY PGQKLIVK YTVKAGESVWGVANKHH SEQID NO:35 7.31 565-607(15) ITMDQLIEWNNIKNN F IYPGQEVIVK YTVKAGESVWGVADSHGSEQ ID NO:36 7.15 623-665 ITMNQLIEWNNIKNN F IYPGQQLIVK Listeria *P60VVVEAGDTLWGIAQSKG SEQ ID NO:37 8.61 30-72(130) 484 P21171 adherencemonocytogenes TTVDAIKKANNLTTD K and invasion IVPGQKLQVN protein P60HAVKSGDTIWALSVKYG SEQ ID NO:38 9.35 203-245 VSVQDIMSWNNLSSS S IYVGQKLAIKinnocua *P60 VVVEAGDTLWGIAQSKG SEQ ID NO:39 8.61 30-72(129) 481 Q01836adherence and TTVDAIKKANNLTTD K invasion IVPGQKLQVN protein P60HNVKSGDTIWALSVKYG SEQ ID NO:40 8.35 201-243 VSVQDIMSWNNLSSS S IYVGQKPAIKivanovii *P60 VVVEAGDTLWGIAQDKG SEQ ID NO:41 6.35 30-72(125) 524 Q01837adherence TTVDALKKANNLTSD K and invasion IVPGQKLQIT protein P60YTVKSGDTIWALSSKYG SEQ ID NO:42 9.37 198-240(73) TSVQNIMSWNNLSSS SIYVGQVLAVK YTVKSGDTLSKIATTFG SEQ ID NO:43 9.89 314-356 TTVSKIKALNGLNSD NLQVGQVLKVK seeligeri *P60 VVVEAGDTLWGIAQDNG SEQ ID NO:44 6.35 30-72(127)523 Q01838 adherence TTVDALKKANKLTTD K and invasion IVPGQKLQVT proteinP60 HTVKSGDTIWALSVKYG SEQ ID NO:45 8.64 200-242(77) ASVQDLMSWNNLSSS SIYVGQNIAVK YTVKSGDTLGKIASTFG SEQ ID NO:46 9.62 320-362 TTVSKIKALNGLTSD NLQVGDVLKVK welshimeri *P60 VVVEAGDTLWGIAQSKG SEQ ID NO:47 8.6130-72(125) 524 M80348 adherence TTVDALKKANNLTSD K and invasionIVPGQKLQVT protein P60 HTVKSGDTIWALSVKYG SEQ ID NO:48 9.35 198-240(75)ASVQDLMSWNNLSSS S IYVGQKIAVK YTVKSGDSLSKIANTFG SEQ ID NO:49 9.89 316-358TSVSKIKALNNLTSD N LQVGTVLKVK grayi *P60 VVVASGDTLWGIASKTG SEQ ID NO:509.42 30-72(104) 511 Q01835 adherence TTVDQLKQLNKLDSD R and invasionIVPGQKLTIK protein P60 YKVKSGDTIWALSVKYG SEQ ID NO:51 9.57 177-219(79)VPVQKLIEWNNLSSS S IYVGQTIAVK YKVQNGDSLGKIASLFK SEQ ID NO:52 8.59 299-342VSVADLTNWNNLNATIT IYAGQELSVK Haemophilus *amiB HIVKKGESLGSLSNKYH SEQ IDNO:53 10.11 294-336 432 P44493 N-acetylmur- influenzae VKVSDIIKLNQLKRK Tamoyl-L- LWLNESIKIP alanine amidase HKVTKNQTLYAISREYN SEQ ID NO:54 10.49387-430 IPVNILLSLNPHLKNG K VITGQKIKLR *yebA YTVTEGDTLKDVLVLSG SEQ IDNO:55 3.87 131-174 475 P44693 homologous to LDDSSVQPLIALDPELAendopeptidase of HLKAGQQFYWI Staphylococcus lppB YKVNKGDTMFLIAYLAG SEQID NO:56 6.40 147-190 405 P44833 outer IDVKELAALNNLSEPNY membraneNLSLGQVLKIS lipoprotein somnus lppB YKVRKGDTMFLIAYISG SEQ ID NO:57 8.56120-164 279 L10653 outer membrane MDIKELATLNNMSEPY lipoproteinHLSIGQVLKIA Helicobacter dniR HVVLPKETLSSIAKRY SEQ ID NO:58 10.02319-361 372 AE000654 regulatory pylori QVSISNIQLANDLKDS N protein DniRIFIHQRLIIR Pseudomonas lppB YIVRRGDTLYSIAFRFG SEQ ID NO:59 10.06 69-113297 P45682 Lipoprotein aeruginosa WDWKALAARNGIAPPYT IQVGQAIQFG PutidanlpD YIVKPGDTLFSIAFRYGW SEQ ID NO:60 9.77 44-87 244 Y19122 lipoproteinDYKELAARNGIPAPYTIR PGQPIRFS Sinohizobium nlpD IMVRQGDTVTVLARRFGV SEQ IDNO:61 10.27 166-209 512 U81296 Lipoprotein meliloti PEKEILKANGLKSASQVEPGQRLVIP Synechocystis nlpD HQVKEGESLWQISQAFQ SEQ ID NO:62 4.38 87-130715 D90915 Lipoprotein sp. VDAKAIALANSISTDTE LQAGQVLNIP slr0878HVVKAGETIDSIAAQYQ SEQ ID NO:63 5.41 4-47 245 D90907 HypotheticalLVPATLISVNNQLSSGQ protein VTPGQTILIP Aqiufex nlpDl YKVKKGDSLWKIAKEYK SEQID NO:64 10.08 26-70(24) 349 AE000700 Lipoprotein aeolicusTSIGKLLELNPKLKNRK YLRPGEKICLK YRVKRGDSLIKIAKKFG SEQ ID NO:65 10.9595-137(37) VSVKEIKRVNKLKGN R IYVGQKLKIP YRVRRGDTLIKIAKRFR SEQ ID NO:6612.11 174-216 TSVKEIKRINRLKGN L IRVGQKLKIP Volvox YTIQPGDTFWAIAQRRG SEQID NO:67 9.03 42-85 309 AF058716 chitinase carteri TTVDVIQSLNPGVVPTRLQVGQVINVP f. nagariensis YTIQPGDTFWAIAQRRG SEQ ID NO:68 9.03 106-149TTVDVIQSLNPGVNPAR LQVGQVINVP Staphylococcus ProtA HVVKPGDTVNDIAKANGSEQIDNO:69 5.58 431-474 524 A04512 protein A aureus TTADKIAADNKLADMIKPGQELVVD lytN YTVKKGDTLSAIALKYK SEQ ID NO:70 10.03 177-220 383 AF106851autolysin TTVSNIQNTNNIANPNL homolog IFIGQKLKVP Colletotrichum cihlHKVKGESLTTIAEKYDT SEQ ID NO:71 4.76 110-153(31) 230 AJ001441glycoprotein GICNIAKLNNLADPNFI DLNQDLQIP lindemuthianumYSVVSGDTLTSIAQALQ SEQ ID NO:72 5.46 185-228 ITLQSLKDANPGVVPEH LNVGQKLNVPChlamydophda amiB IVYREGDSLSKIAKKYK SEQ ID NO:73 9.46 159-201 205AE001659 N-Acetylmur- LSVTELKKINKLDSD A .1 amoyl-L-Ala IYAGQRLCLQAmidase Pneumoniae CPn0593 YVVQDGDSLWLIAKRFG SEQ ID NO:74 10.01 316-358362 AE001643 IPMDKIIQKNGLNHH R LFPGKVLKLP NlpD VVVKKGDFLERIARANH SEQ IDNO:75 10.17 124-166 233 AE001670 Muramidase TTVAKLMQINDLTTT Q LKIGQVIKVPYIVQEGDSPWTIALRNHI SEQ ID NO:76 8.64 188-233 RLDDLLKMNDLDEYKARRLKPGDQLFIR Chlamydia NlpD VIVKKGDFLERIARSNH SEQ ID NO:77 9.99 138-180245 AE001348 Muramidase trachomatis TTVSALMQLNDLSST Q LQIGQVLRVPYVVKEGDSPWAIALSNG SEQ ID NO:78 10.00 200-245 IRLDELLKLNGLDEQKARRLRPGDRLRIR papQ HIVKQGETLSKIASKYN SEQ ID NO:79 9.89 155-197 200AE001330 IPVVELKKLNKLNSD T IFTDQRIRLP Prevotella phg HTVRSNESLYDISQQYGSEQ ID NO:80 10.72 266-309 309 AF017417 hemagglutinin intermediaVRLKNIMKANRKIVKRGI KAGDRVVL Leuconostoc lys YTVQSGDTLGAIAAKYG SEQ IDNO:81 9.23 335-378 432 endolysin oenos 10MC TTYQKIASLNGIGSPYIIIPGEKLKVS YKVASGDTLSAIASKYG SEQ ID NO:82 9.68 389-432TSVSKLVSLNGLKNANY IYVGENLKIK Oenococcus Lys44 YTVRSGDTLGAIAAKYG SEQ IDNO:83 9.58 335-378 432 AF047001 Lysin oeni fOg44 TTYQKLASLNGIGSPYIIIPGEKLKVS YKVASGDTLSAIASKYG SEQ ID NO:84 9.95 389-432 TSVSKLVSLNGLKNANYIYVGQTLRIK Thermotoga TM0409 YKVQKNDTLYSISLNFG SEQ ID NO:85 5.49 26-69271 AE001720 maritime ISPSLLLDWNPGLDPHS LRVGQEIVIP YTVKKGDTLDAIAKRFF SEQID NO:86 9.73 76-118 TTATFIKEANQLKSY T IYAGQKLFIP TM1686HVVKRGETLWSIANQYG SEQ ID NO:87 8.76 212-255 395 AE001809VRVGDIVLINRLEDPDR IVAGQVLKIG Treponema dniR HTIRSGDTLYALARRYG SEQ IDNO:88 10.58 607-650 779 AE001237 membrane- pallidum LGVDTLKAHNRAHSATHbound lytic LKIGQKLIIP murein trans- glycosylase D HVVQQGDTLWSLAKRYGVSEQ ID NO:89 4.81 734-777 SVENLAEENNLAVDATLS LGMILKTP TP0155YEVREGDVVGRIAQRYD SEQ ID NO:90 9.58 87-130 371 AE001200ISQDAIISLNKLRSTRA LQVGQLLKIP TP0444 HVIAKGETLFSLSRRYG SEQ ID NO:91 10.9867-110 342 AE001221 VPLSALAQANNLANVHQ LVPGQRIVVP Borrelia BB0262HKIKPGETLSHVAARYQ SEQ ID NO:92 9.72 183-226(6) 417 AE001137 Hypotheticalburgdorferi ITSETLISFNEIKDVRN protein IKPNSVIKVP YIVKKNDSISSIASAYN SEQID NO:93 4.58 233-275 VPKVDILDSNNLDNE V LFLGQKLFIP *BB0625YKVVKGDTLFSIAIKYK SEQ ID NO:94 10.02 44-86(28) 697 AE001164 N-acetylmur-VKVSDLKRINKLNVD N amoyl-L- IKAGQILIIP alanine amidase YTAKEGDTIESISKLVGSEQ ID NO:95 5.00 115-157(7) LSQEEIIAWNDLRSK D LKVGMKLVLTYMVRKGDSLSKLSQDFD SEQ ID NO:96 9.20 165-207(8) ISSKDILKFNFLNDD KLKIGQQLFLK HYVKRGETLGRIAYIYG SEQ ID NO:97 10.05 216-258(27)VTAKDLVALNGNRAI N LKAGSLLNVL HSVAVGETLYSIARHYG SEQ ID NO:98 7.41 286-328VLIEDLKNWNNLSSN N IMHDQKLKIF BB0761 YKVKKGDTFTKIANKIN SEQ ID NO:99 9.3459-102 295 AE001176 GWQSGIATINLLDSP A VSVGQEILIP Lacrobacillus *lysYTVVSGDSWNKIAQRNGL SEQ ID NO:100 9.83 399-442 442 X90511 lysin gleSMYTLASQNGKSIYSTI YPGNKLIIK Bacillus *lytE IKVKKGDTLWDLSRKYD SEQ IDNO:101 9.55 28-70(17) 334 U38819.1 D-Glutamate- subtilis TTISKIKSENHLRSDI M-diaminopim- IYVGQTLSIN late endo- peptidase YKVKSGDSLWKISKKYG SEQ IDNO:102 10.16 88-130(20) MTINELKKLNGLKSD L LRVGQVLKLK YKVKSGDSLSKIASKYGSEQ ID NO:103 10.03 151-193 TTVSKLKSLNGLKSD V IYVNQVLKVK spoVIDCIVQQEDTIERLCERYEI SEQ ID NO:104 4.20 525-568 575 P37963 stage VI spor-TSQQLIRMNSLALDDELK ulation pro- AGQILYIP tein D yaaH MVKQGDTLSAIASQYRTSEQ ID NO:105 3.89 1-43(5) 427 P37531 Hypothetical TTNDITETNEIPNPDSLprotein VVGQTIVIP YDVKRGDTLTSIARQFN SEQ ID NO:106 10.32 49-92TTAAELARVNRIQLNTV LQIGFRLYIP yhdD IKVKSGDSLWKLAQTYN SEQ ID NO:107 9.5629-71(22) 488 Y14079 Hypothetical TSVAALTSANHLSTT V protein LSIGQTLTIPYTVKSGDSLWLIANEFM SEQ ID NO:108 9.62 94-136(39) TVQELKKLNGLSSD LIRAGQKLKVS YKVQLGDSLWKIANKVN SEQ ID NO:109 9.72 176-218(23)MSIAELKVLNNLKSD T IYVNQVLKTK YTVKSGDSLWKIANNYN SEQ ID NO:110 9.65242-284(24) LTVQQIRNINNLKSD V LYVGQVLKLT YTVSGDSLWVIAQKFNV SEQ ID NO:1119.72 309-351 TAQQIREKNNLKTD VL GVGQKLVIS yojL IKVKSGDSLWKLSRQYD SEQ IDNO:112 9.93 29-71(18) 414 Z99114 similar to TTISALKSENKLKST V cell wallLYVGQSLKVP binding protein YTVAYGDSLWMIAKNHM SEQ ID NO:113 9.8190-132(26) SVSELKSLNSLSSD LI RPGQKLKIK YTVKLGDSLWKIANSLN SEQ ID NO:1149.27 159-201(25) MTVAELKTLNGLTSD T LYPKQVLKIG YKVKAGDSLWKIANRLG SEQ IDNO:115 9.84 227-269 VTVQSIRDKNNLSSD V LQIGQVLTIS yocH ITVQKGDTLWGISQKNGSEQ ID NO:116 9.25 28-70(9) 287 AF027868 similar KGVNLKDLKEWNKLTSD K topapQ IIAGEKLTIS YTIKAGDTLSKIAQKFG SEQ ID NO:117 9.64 80-122TTVNNLKVWNNLSSD M IYAGSTLSVK ykvP HHVTPGETLSIIASKYN SEQ ID NO:118 8.65345-387 399 Z99111 Hypothetical VSLQQLNELNHFKSD Q protein IYAGQIIKIR*xlyB YHVKKGDTLSGIAASEG SEQ ID NO:119 9.72 179-222 317 Z99110N-acetylmur- ASVKTLQSINHITDPNH amoyl-L- IKIGQVIKLP alanine amidase yrbAHIVQKGDSLWKIAEKYG SEQ ID NO:120 8.51 4-48 387 Z99118 similar toVDVEEVKKLNTQLSNPD spore coat LIMPGMKIKVP protein ydhD HIVGPGDSLFSIGRRYGSEQ ID NO:121 5.49 4-46 439 Z99107 ASVDQIRGVNGLDET N IVPGQALLIP ykuDYQVKQGDTLNSIAADFR SEQ ID NO:122 6.10 4-46 164 Z99111 ISTAALLQANPSLQA GLTAGQSIVIP PBSX *xlyA YVVKQGDTLTSIAPAFG SEQ ID NO:123 4.65 161-204 297P39800 N-acetylmur- VTVAQLQEWNNIEDPNL amoyl-L- IRVGQVLIVS alanineamidase PZA *orf15 YKVKSGDNLTKIAKHN SEQ ID NO:124 10.23 163-207(6) 258P11187 TTVATLLKLNPSIKDP NMIRVGQTINVT (=.29) HKVKSGDTLSKIAVDNK SEQ IDNO:125 10.17 214-258 P07540 TTVSRLMSLNPEITNPN HIKVGQTIRLS B103 *orfL5HVVKKGDTLSEIAKKI SEQ ID NO:126 10.11 165-209(9) 263 X99260 lysozymeKTSTKTLLELNPTIKN PNKIYVGQRINVG YKIKRGETLTGIAKKT SEQ ID NO:127 10.61219-263 TVSQLMKLNPNIKAIN NYAGQTIRLK sphaericus *Pep1 ILIRPGDSLWYFSDLFKSEQ ID NO:128 8.86 3-46(6) 396 X69507 carboxypepti- IPLQLLLDSNRNINPQ LLQVGQRIQIP dase I YTITQGDSLWQIAQNKN SEQ ID NO:129 7.15 53-96LPLNAILLVNPEIQPS R LHIGQTIQVP Salmonella nlpD YTVKKGDTLFYIAWITG SEQ IDNO:130 8.64 121-164 377 AJ006131 dublin NDFRDLAQRNSISAPY SLNVGQTLQVGEscherichia *yebA YVVSTGDTLSSILNQYG SEQ ID NO:131 4.13 77-121 419 p24204homologous to coli IDMGDISQLAAADKELR endopeptidase NLKIGQQLSWT ofStaphy- lococcus mltD YTVRSGDTLSSIASRLG SEQ ID NO:132 10.58 343-385(16)452 P23931 MEMBRANE- VSTKDLQQWNKLRGS K BOUND LYTIC LKPGQSLTIG MUREINTRANS- GLYCOSYLASE D PRECURSOR YRVRKGDSLSSIAKRHG SEQ ID NO:133 10.18402-443 VNIKDVMRWNSDTAN L QPGDKLTLF UUG YTVKRGDTLYRISRTTG SEQ ID NO:13410.16 50-93 259 U28375 Hypothetical TSVKELARLNGISPPYT protein IEVGQKLKLGnlpD YTVKKGDTLFYIAWITG SEQ ID NO:135 8.64 123-166 379 P33648 LipoproteinNDFRLAQRNNIQAPYAL NVGQTLQVG Drosophila Q9VNA1 YTVGNRDTLTSVAA SEQ IDNO:136 7.15 329-371 1325 AF125384 Lethal S2FD melanogasterRFDTTPSELTHLNR protein LNSS FIYPGQQLLVP Drosophila Q961P8YTVGNRDTLTSVAARFD SEQ ID NO:136 7.15 104-146 678 AAK92873 melanogasterTTPSELTHLNRLNSS F IYPGQQLLVP Caenorhabditis F43G9.2 RKVKUGDTLNKLAIKYQSEQ ID NO:137 10.01 12-55 179 Z79755 elegans VNVAEIKRVNNMVSEQDFMALSKVKIP Caenorhabditis F52E1.13 YTITETDTLERVAASHD SEQ ID NO:138 7.0824-66 819 U41109 elegans CTVGELMKLNKMASR M VFPGQKILVP CaenorhabditisF07G11.9 TEIKSGDSCWNIASNAK SEQ ID NO:139 8.32 23-66(11) 1614 U64836/Putative elegans ISVERLQQLNKGMKCDK LPLGDKLCLA AF016419 EndochitinaseLKLKAEDTCFKIWSSQK SEQ ID NO:140 7.84 78-121(21) LSERQFLGMNEGMDCDKLKVGKEVCVA HKIQKGDTCFKIWTTNK SEQ ID NO:141 8.65 143-186(21)ISEKQFRNLNKGLDCDK LEIGKEVCIS LKIKEGDTCYNIWTSQK SEQ ID NO:142 4.54208-251(19) ISEQEFMELNKGLDCDK LEIGKEVCVT YRFKKGDTCYKIWTSHK SEQ ID NO:1439.35 271-314(20) MSEKQFRALNRGIDCDR LVPGKELCVG ITVKPGDTCFSIWTSQK SEQ IDNO:144 4.21 335-378(23) MTQQQFMDINPELDCDK LEIGKEVCVT VKINPGDTCFNIWTSQRSEQ ID NO:145 6.30 402-445(21) NTQQQFMDLNKRLDCDK LEVGKEVCVAVQINPGDTCFKIWSAQK SEQ ID NO:146 4.60 467-510(37) LTEQQFMELNKGLDCDRLEVGKEVCIA TEVKEGDTCFKIWSAHK SEQ ID NO:147 5.12 548-591(44)ITEQQFMEMNRGLDCNR LEVGKEVCIV IKVKEGDTCFKIWSAQK SEQ ID NO:148 7.85636-679(66) MTEQQFMEMNRGLDCNK LMVGKEVCVS ATITPGNTCFNISVAYG SEQ ID NO:1493.99 746-786(8) INLT DLQKTYDCKALE VGDTICVS IEVIKGDTCWFLENAFK SEQ IDNO:150 4.67 795-838 TNQTEMERANEGVKCDN LPIGRMMCVW Caenorhabditis T01C4.1HTIKSGDTCWKIASEA SEQ ID NO:151 5.01 23-66(51) 1484 U70858 Putativeelegans SISVQELEGLNSKKSC Endochitinase ANLAVGLSEQEF IHVKEGDTCYTIWTSQHSEQ ID NO:152 4.12 118-161(25) LTEKQFMDMNEELNCGM LEIGNEVCVDATVTPGSSCYTISASYG SEQ ID NO:153 3.07 187-226(9) LNLAELQTTYNCDALQVDDTICVS IEILNGDTCGFLENAFQ SEQ ID NO:154 3.85 236-279 TNNTEMEIANEGVKCDNLPIGRMMCVW Bacillus #ypbE HTVQKKETLYRISMKYY SEQ ID NO:155 9.45 191-136240 L47648 subtilis KSRTGEEKIRAYNHLNG NDVYTGQVLDIP Citrobacter #eaeYTLKTGESVAQLSKSQG SEQ ID NO:156 8.59 65-113 936 Q07591 fruendiiISVPVIWSLNKHLYSSE SEMMKASPGQQIILP Escherichia #eae YTLKTGETVADLSKSQD SEQID NO:157 5.65 65-113 934 P43261 Necessary coli INLSTIWSLNKHLYSSE forclose SEMMKAAPQQIILP (intimate) attachment of bacteria Micrococcus #rpfIVVKSGDSLWTLANEYE SEQ ID NO:158 3.85 171-218 220 Z96935 Bacterial luteusVEGGWTALYEANKGAVS Cytokine DAAVIYVGQELVL Bacillus #yneA IEVQQGDTLWSIADQSEQ ID NO:159 3.81 40-90 105 Z73234 subtilis VADTKKINDKNDFIEWVADKNQLQTSDIQPGDEL VIP Streptococcus # YTVKYGDTLSTIAEAMG SEQ ID NO:1604.23 47-103 393 U09352 pyogenes IDVHVLGDINHIANIDL IFPDTILTANYNQHGQA TTLTBacillus #xkdP YTVKKGDTLWDIAGRFY SEQ ID NO:161 11.23 176-234 235 p54335subtilis GNSTQWRKIWNANKTAM IKRSKRNIRQPGHWIFP GQKLKIP Bacillus #yqbPYTVKKGDTLWDIAGRFY SEQ ID NO:161 11.23 177-234 235 G1225954 subtilisGNSTQWRKIWNANKTAM IKRSKRNIRQPGHWIFP GQKLKIP Bacillus # YTVKKGDTLWDLAGKFYSEQ ID NO:162 10.75 161-218 219 P45932 subtilis GDSTKWRKIWKVNKKANIKRSKRNIRQPGHWIF PGQKLKIP a)Proteins listed were obtained by a homologysearch in the SWISSPROT, PIR, and Genbank databases with the repeats ofAcmA using the BLAST program. b)*genes encoding cell wall hydrolases.#proteins containing repeats that are longer than the con- sensussequence. c)The number of aa residues between the repeats are givenbetween brackets. d)Number of aa of the primary translation product.e)Genbank accession number. Consensus repeatYxVKxGDTLxxIAxxxxxxxxxLxxxNxxLxxxxxIxxGQxIxVx (SEQ ID NO:163)                 H IR  ESV  LS         I      I     L     L I                     L     I  V                       V     V L

[0164] TABLE 4 Calculated pI's of individual repeat sequences of theAcmA and AcmD protein anchors. AcmA anchor domain AcmD anchor domainCalculated Calculated Repeat pI Repeat pI A1 9.75 D1 4.15 A2 9.81 D23.78 A3 10.02 D3 4.15 A1A2A3 10.03 D1D2D3 3.85

[0165] TABLE 5 Hybrid protein anchors composed of different AcmA andAcmD repeat sequences and their calculated pI's. Composition of hybridsCalculated AcmA-repeat sequence AcmD-repeat sequence pI A1A2A3 — 10.03A1A2 D1 9.53 A1A2A3 D1D2D3 8.66 A1 D2 8.45 A3 D1D2 7.39 A1A2 D1D2D3 6.08A3 D1D2D3 5.07 A1 D1D2D3 4.37 — D1D2D3 3.85

[0166]

1 165 1 5 PRT Artificial Sequence SITE (1)..(5) motif, anchoringportion, “Xaa” can be any amino acid 1 Leu Pro Xaa Thr Gly 1 5 2 30 DNAArtificial Sequence synthesized sequence, primer MSA2.1 2 accatggcaaaaaatgaaag taaatatagc 30 3 48 DNA Artificial Sequence synthesizedsequence, primer MSA2.4 3 cggtctctag cttataagct tagaattcgg gatgttgctgctccacag 48 4 32 DNA Artificial Sequence synthesized sequence, primerPrtP.sspro.fw 4 ccgtctccca tgcaaaggaa aaaagaaagg gc 32 5 46 DNAArtificial Sequence synthesized sequence, primer PrtP.sspro.rev 5aaaaaaagct tgaattccca tggcagtcgg ataataaact ttcgcc 46 6 44 DNAArtificial Sequence synthesized sequence, primer pACMB2 6 cgcaagcttctgcagagctc ttagattcta attgtttgtc ctgg 44 7 30 DNA Artificial Sequencesynthesized sequence, primer pACMB3 7 cggaattcaa ggaggagaaa tatcaggagg30 8 31 DNA Artificial Sequence synthesized sequence, primerPrtP.cwa.fw3 8 atataaagct tgcaaagtct gaaaacgaag g 31 9 35 DNA ArtificialSequence synthesized sequence, primer PrtP.cwa.rev 9 ccgtctcaagctcactattc ttcacgttgt ttccg 35 10 35 DNA Artificial Sequence synthesizedsequence, primer cArepeat3.fw 10 ccgtctccaa ttcaatctgc tgctgcttca aatcc35 11 58 DNA Artificial Sequence synthesized sequence, primercArepeat3.rev 11 taataagctt aaaggtctcc aattcctttt attcgtagat actgaccaattaaaatag 58 12 38 DNA Artificial Sequence synthesized sequence, primercDrepeat1.fw 12 ccgtctccaa tttcaggagg aactgctgtt acaactag 38 13 50 DNAArtificial Sequence synthesized sequence, primer cDrepeat3.rev 13taataagctt aaaggtctcc aattccagca acttgcaaaa cttctcctac 50 14 37 DNAArtificial Sequence synthesized sequence, primer ppmA.1 14 cggtctcacatgtcgaaagg gtcagaaggt gcagacc 37 15 39 DNA Artificial Sequencesynthesized sequence, primer ppmA.2 15 cggtctcgaa ttgcttcgtt tgatgtactactgcttgag 39 16 43 PRT Lactococcus lactis SITE (1)..(43) AcmA cell wallbinding domain homologue 16 Tyr Thr Val Lys Ser Gly Asp Thr Leu Trp GlyIle Ser Gln Arg Tyr 1 5 10 15 Gly Ile Ser Val Ala Gln Ile Gln Ser AlaAsn Asn Leu Lys Ser Thr 20 25 30 Ile Ile Tyr Ile Gly Gln Lys Leu Val LeuThr 35 40 17 43 PRT Lactococcus lactis SITE (1)..(43) AcmA cell wallbinding domain homologue 17 Val Lys Val Lys Ser Gly Asp Thr Leu Trp AlaLeu Ser Val Lys Tyr 1 5 10 15 Lys Thr Ser Ile Ala Gln Leu Lys Ser TrpAsn His Leu Ser Ser Asp 20 25 30 Thr Ile Tyr Ile Gly Gln Asn Leu Ile ValSer 35 40 18 43 PRT Lactococcus lactis SITE (1)..(43) AcmA cell wallbinding domain homologue 18 His Lys Val Val Lys Gly Asp Thr Leu Trp GlyLeu Ser Gln Lys Ser 1 5 10 15 Gly Ser Pro Ile Ala Ser Ile Lys Ala TrpAsn His Leu Ser Ser Asp 20 25 30 Thr Ile Leu Ile Gly Gln Tyr Leu Arg IleLys 35 40 19 44 PRT Lactococcus lactis SITE (1)..(44) AcmA cell wallbinding domain homologue 19 Tyr Lys Val Gln Glu Gly Asp Ser Leu Ser AlaIle Ala Ala Gln Tyr 1 5 10 15 Gly Thr Thr Val Asp Ala Leu Val Ser AlaAsn Ser Leu Glu Asn Ala 20 25 30 Asn Asp Ile His Val Gly Glu Val Leu GlnVal Ala 35 40 20 46 PRT Lactococcus lactis SITE (1)..(46) AcmA cell wallbinding domain homologue 20 Tyr Thr Val Lys Ser Gly Asp Ser Leu Tyr SerIle Ala Glu Gln Tyr 1 5 10 15 Gly Met Thr Val Ser Ser Leu Met Ser AlaAsn Gly Ile Tyr Asp Val 20 25 30 Asn Ser Met Leu Gln Val Gly Gln Val LeuGln Val Thr Val 35 40 45 21 43 PRT Lactococcus lactis SITE (1)..(43)AcmA cell wall binding domain homologue 21 Tyr Thr Ile Gln Asn Gly AspSer Ile Tyr Ser Ile Ala Thr Ala Asn 1 5 10 15 Gly Met Thr Ala Asp GlnLeu Ala Ala Leu Asn Gly Phe Gly Ile Asn 20 25 30 Asp Met Ile His Pro GlyGln Thr Ile Arg Ile 35 40 22 44 PRT Lactococcus bacteriophage Tuc2009SITE (1)..(44) AcmA cell wall binding domain homologue 22 Tyr Val ValLys Gln Gly Asp Thr Leu Ser Gly Ile Ala Ser Asn Trp 1 5 10 15 Gly ThrAsn Trp Gln Glu Leu Ala Arg Gln Asn Ser Leu Ser Asn Pro 20 25 30 Asn MetIle Tyr Ala Gly Gln Val Ile Ser Phe Thr 35 40 23 43 PRT Lactococcusbacteriophage Tuc2009 SITE (1)..(43) AcmA cell wall binding domainhomologue 23 Tyr Thr Val Gln Ser Gly Asp Asn Leu Ser Ser Ile Ala Ile LeuLeu 1 5 10 15 Gly Thr Thr Val Gln Ser Leu Val Ser Met Asn Gly Ile SerAsn Pro 20 25 30 Asn Leu Ile Tyr Ala Gly Gln Thr Leu Asn Tyr 35 40 24 44PRT Lactococcus bacteriophage LC3 SITE (1)..(44) AcmA cell wall bindingdomain homologue 24 Tyr Ile Val Lys Gln Gly Asp Thr Leu Ser Gly Ile AlaSer Asn Leu 1 5 10 15 Gly Thr Asn Trp Gln Glu Leu Ala Arg Gln Asn SerLeu Ser Asn Pro 20 25 30 Asn Met Ile Tyr Ser Gly Gln Val Ile Ser Leu Thr35 40 25 43 PRT Lactococcus bacteriophage LC3 SITE (1)..(43) AcmA cellwall binding domain homologue 25 Tyr Thr Val Gln Ser Gly Asp Asn Leu SerSer Ile Ala Arg Arg Leu 1 5 10 15 Gly Thr Thr Val Gln Ser Leu Val SerMet Asn Gly Ile Ser Asn Pro 20 25 30 Asn Leu Ile Tyr Ala Gly Gln Thr LeuAsn Tyr 35 40 26 43 PRT Enterococcus faecalis SITE (1)..(43) AcmA cellwall binding domain homologue 26 Tyr Thr Val Lys Ser Gly Asp Thr Leu AsnLys Ile Ala Ala Gln Tyr 1 5 10 15 Gly Val Ser Val Ala Asn Leu Arg SerTrp Asn Gly Ile Ser Gly Asp 20 25 30 Leu Ile Phe Val Gly Gln Lys Leu IleVal Lys 35 40 27 43 PRT Enterococcus faecalis SITE (1)..(43) AcmA cellwall binding domain homologue 27 Tyr Thr Val Lys Ser Gly Asp Thr Leu AsnLys Ile Ala Ala Gln Tyr 1 5 10 15 Gly Val Thr Val Ala Asn Leu Arg SerTrp Asn Gly Ile Ser Gly Asp 20 25 30 Leu Ile Phe Val Gly Gln Lys Leu IleVal Lys 35 40 28 43 PRT Enterococcus faecalis SITE (1)..(43) AcmA cellwall binding domain homologue 28 Tyr Thr Ile Lys Ser Gly Asp Thr Leu AsnLys Ile Ala Ala Gln Tyr 1 5 10 15 Gly Val Ser Val Ala Asn Leu Arg SerTrp Asn Gly Ile Ser Gly Asp 20 25 30 Leu Ile Phe Ala Gly Gln Lys Ile IleVal Lys 35 40 29 43 PRT Enterococcus faecalis SITE (1)..(43) AcmA cellwall binding domain homologue 29 Tyr Thr Ile Lys Ser Gly Asp Thr Leu AsnLys Ile Ser Ala Gln Phe 1 5 10 15 Gly Val Ser Val Ala Asn Leu Arg SerTrp Asn Gly Ile Lys Gly Asp 20 25 30 Leu Ile Phe Ala Gly Gln Thr Ile IleVal Lys 35 40 30 43 PRT Enterococcus faecalis SITE (1)..(43) AcmA cellwall binding domain homologue 30 His Thr Val Lys Ser Gly Asp Ser Leu TrpGly Leu Ser Met Gln Tyr 1 5 10 15 Gly Ile Ser Ile Gln Lys Ile Lys GlnLeu Asn Gly Leu Ser Gly Asp 20 25 30 Thr Ile Tyr Ile Gly Gln Thr Leu LysVal Gly 35 40 31 43 PRT Enterococcus hirae SITE (1)..(43) AcmA cell wallbinding domain homologue 31 Tyr Thr Val Lys Ser Gly Asp Ser Val Trp GlyIle Ser His Ser Phe 1 5 10 15 Gly Ile Thr Met Ala Gln Leu Ile Glu TrpAsn Asn Ile Lys Asn Asn 20 25 30 Phe Ile Tyr Pro Gly Gln Lys Leu Thr IleLys 35 40 32 43 PRT Enterococcus hirae SITE (1)..(43) AcmA cell wallbinding domain homologue 32 Tyr Thr Val Lys Ser Gly Asp Ser Val Trp LysIle Ala Asn Asp His 1 5 10 15 Gly Ile Ser Met Asn Gln Leu Ile Glu TrpAsn Asn Ile Lys Asn Asn 20 25 30 Phe Val Tyr Pro Gly Gln Gln Leu Val ValSer 35 40 33 43 PRT Enterococcus hirae SITE (1)..(43) AcmA cell wallbinding domain homologue 33 Tyr Thr Val Lys Ala Gly Glu Ser Val Trp SerVal Ser Asn Lys Phe 1 5 10 15 Gly Ile Ser Met Asn Gln Leu Ile Gln TrpAsn Asn Ile Lys Asn Asn 20 25 30 Phe Ile Tyr Pro Gly Gln Lys Leu Ile ValLys 35 40 34 43 PRT Enterococcus hirae SITE (1)..(43) AcmA cell wallbinding domain homologue 34 Tyr Thr Val Lys Ala Gly Glu Ser Val Trp GlyVal Ala Asn Lys Asn 1 5 10 15 Gly Ile Ser Met Asn Gln Leu Ile Glu TrpAsn Asn Ile Lys Asn Asn 20 25 30 Phe Ile Tyr Pro Gly Gln Lys Leu Ile ValLys 35 40 35 43 PRT Enterococcus hirae SITE (1)..(43) AcmA cell wallbinding domain homologue 35 Tyr Thr Val Lys Ala Gly Glu Ser Val Trp GlyVal Ala Asn Lys His 1 5 10 15 His Ile Thr Met Asp Gln Leu Ile Glu TrpAsn Asn Ile Lys Asn Asn 20 25 30 Phe Ile Tyr Pro Gly Gln Glu Val Ile ValLys 35 40 36 43 PRT Enterococcus hirae SITE (1)..(43) AcmA cell wallbinding domain homologue 36 Tyr Thr Val Lys Ala Gly Glu Ser Val Trp GlyVal Ala Asp Ser His 1 5 10 15 Gly Ile Thr Met Asn Gln Leu Ile Glu TrpAsn Asn Ile Lys Asn Asn 20 25 30 Phe Ile Tyr Pro Gly Gln Gln Leu Ile ValLys 35 40 37 43 PRT Listeria monocytogenes SITE (1)..(43) AcmA cell wallbinding domain homologue 37 Val Val Val Glu Ala Gly Asp Thr Leu Trp GlyIle Ala Gln Ser Lys 1 5 10 15 Gly Thr Thr Val Asp Ala Ile Lys Lys AlaAsn Asn Leu Thr Thr Asp 20 25 30 Lys Ile Val Pro Gly Gln Lys Leu Gln ValAsn 35 40 38 43 PRT Listeria monocytogenes SITE (1)..(43) AcmA cell wallbinding domain homologue 38 His Ala Val Lys Ser Gly Asp Thr Ile Trp AlaLeu Ser Val Lys Tyr 1 5 10 15 Gly Val Ser Val Gln Asp Ile Met Ser TrpAsn Asn Leu Ser Ser Ser 20 25 30 Ser Ile Tyr Val Gly Gln Lys Leu Ala IleLys 35 40 39 43 PRT Listeria monocytogenes SITE (1)..(43) AcmA cell wallbinding domain homologue 39 Val Val Val Glu Ala Gly Asp Thr Leu Trp GlyIle Ala Gln Ser Lys 1 5 10 15 Gly Thr Thr Val Asp Ala Ile Lys Lys AlaAsn Asn Leu Thr Thr Asp 20 25 30 Lys Ile Val Pro Gly Gln Lys Leu Gln ValAsn 35 40 40 43 PRT Listeria innocua SITE (1)..(43) AcmA cell wallbinding domain homologue 40 His Asn Val Lys Ser Gly Asp Thr Ile Trp AlaLeu Ser Val Lys Tyr 1 5 10 15 Gly Val Ser Val Gln Asp Ile Met Ser TrpAsn Asn Leu Ser Ser Ser 20 25 30 Ser Ile Tyr Val Gly Gln Lys Pro Ala IleLys 35 40 41 43 PRT Listeria ivanovii SITE (1)..(43) AcmA cell wallbinding domain homologue 41 Val Val Val Glu Ala Gly Asp Thr Leu Trp GlyIle Ala Gln Asp Lys 1 5 10 15 Gly Thr Thr Val Asp Ala Leu Lys Lys AlaAsn Asn Leu Thr Ser Asp 20 25 30 Lys Ile Val Pro Gly Gln Lys Leu Gln IleThr 35 40 42 43 PRT Listeria ivanovii SITE (1)..(43) AcmA cell wallbinding domain homologue 42 Tyr Thr Val Lys Ser Gly Asp Thr Ile Trp AlaLeu Ser Ser Lys Tyr 1 5 10 15 Gly Thr Ser Val Gln Asn Ile Met Ser TrpAsn Asn Leu Ser Ser Ser 20 25 30 Ser Ile Tyr Val Gly Gln Val Leu Ala ValLys 35 40 43 43 PRT Listeria ivanovii SITE (1)..(43) AcmA cell wallbinding domain homologue 43 Tyr Thr Val Lys Ser Gly Asp Thr Leu Ser LysIle Ala Thr Thr Phe 1 5 10 15 Gly Thr Thr Val Ser Lys Ile Lys Ala LeuAsn Gly Leu Asn Ser Asp 20 25 30 Asn Leu Gln Val Gly Gln Val Leu Lys ValLys 35 40 44 43 PRT Listeria seeligeri SITE (1)..(43) AcmA cell wallbinding domain homologue 44 Val Val Val Glu Ala Gly Asp Thr Leu Trp GlyIle Ala Gln Asp Asn 1 5 10 15 Gly Thr Thr Val Asp Ala Leu Lys Lys AlaAsn Lys Leu Thr Thr Asp 20 25 30 Lys Ile Val Pro Gly Gln Lys Leu Gln ValThr 35 40 45 43 PRT Listeria seeligeri SITE (1)..(43) AcmA cell wallbinding domain homologue 45 His Thr Val Lys Ser Gly Asp Thr Ile Trp AlaLeu Ser Val Lys Tyr 1 5 10 15 Gly Ala Ser Val Gln Asp Leu Met Ser TrpAsn Asn Leu Ser Ser Ser 20 25 30 Ser Ile Tyr Val Gly Gln Asn Ile Ala ValLys 35 40 46 43 PRT Listeria seeligeri SITE (1)..(43) AcmA cell wallbinding domain homologue 46 Tyr Thr Val Lys Ser Gly Asp Thr Leu Gly LysIle Ala Ser Thr Phe 1 5 10 15 Gly Thr Thr Val Ser Lys Ile Lys Ala LeuAsn Gly Leu Thr Ser Asp 20 25 30 Asn Leu Gln Val Gly Asp Val Leu Lys ValLys 35 40 47 43 PRT Listeria welshimeri SITE (1)..(43) AcmA cell wallbinding domain homologue 47 Val Val Val Glu Ala Gly Asp Thr Leu Trp GlyIle Ala Gln Ser Lys 1 5 10 15 Gly Thr Thr Val Asp Ala Leu Lys Lys AlaAsn Asn Leu Thr Ser Asp 20 25 30 Lys Ile Val Pro Gly Gln Lys Leu Gln ValThr 35 40 48 43 PRT Listeria welshimeri SITE (1)..(43) AcmA cell wallbinding domain homologue 48 His Thr Val Lys Ser Gly Asp Thr Ile Trp AlaLeu Ser Val Lys Tyr 1 5 10 15 Gly Ala Ser Val Gln Asp Leu Met Ser TrpAsn Asn Leu Ser Ser Ser 20 25 30 Ser Ile Tyr Val Gly Gln Lys Ile Ala ValLys 35 40 49 43 PRT Listeria welshimeri SITE (1)..(43) AcmA cell wallbinding domain homologue 49 Tyr Thr Val Lys Ser Gly Asp Ser Leu Ser LysIle Ala Asn Thr Phe 1 5 10 15 Gly Thr Ser Val Ser Lys Ile Lys Ala LeuAsn Asn Leu Thr Ser Asp 20 25 30 Asn Leu Gln Val Gly Thr Val Leu Lys ValLys 35 40 50 43 PRT Listeria grayi SITE (1)..(43) AcmA cell wall bindingdomain homologue 50 Val Val Val Ala Ser Gly Asp Thr Leu Trp Gly Ile AlaSer Lys Thr 1 5 10 15 Gly Thr Thr Val Asp Gln Leu Lys Gln Leu Asn LysLeu Asp Ser Asp 20 25 30 Arg Ile Val Pro Gly Gln Lys Leu Thr Ile Lys 3540 51 43 PRT Listeria grayi SITE (1)..(43) AcmA cell wall binding domainhomologue 51 Tyr Lys Val Lys Ser Gly Asp Thr Ile Trp Ala Leu Ser Val LysTyr 1 5 10 15 Gly Val Pro Val Gln Lys Leu Ile Glu Trp Asn Asn Leu SerSer Ser 20 25 30 Ser Ile Tyr Val Gly Gln Thr Ile Ala Val Lys 35 40 52 44PRT Listeria grayi SITE (1)..(44) AcmA cell wall binding domainhomologue 52 Tyr Lys Val Gln Asn Gly Asp Ser Leu Gly Lys Ile Ala Ser LeuPhe 1 5 10 15 Lys Val Ser Val Ala Asp Leu Thr Asn Trp Asn Asn Leu AsnAla Thr 20 25 30 Ile Thr Ile Tyr Ala Gly Gln Glu Leu Ser Val Lys 35 4053 43 PRT Haemophilus influenzae SITE (1)..(43) AcmA cell wall bindingdomain homologue 53 His Ile Val Lys Lys Gly Glu Ser Leu Gly Ser Leu SerAsn Lys Tyr 1 5 10 15 His Val Lys Val Ser Asp Ile Ile Lys Leu Asn GlnLeu Lys Arg Lys 20 25 30 Thr Leu Trp Leu Asn Glu Ser Ile Lys Ile Pro 3540 54 44 PRT Haemophilus influenzae SITE (1)..(44) AcmA cell wallbinding domain homologue 54 His Lys Val Thr Lys Asn Gln Thr Leu Tyr AlaIle Ser Arg Glu Tyr 1 5 10 15 Asn Ile Pro Val Asn Ile Leu Leu Ser LeuAsn Pro His Leu Lys Asn 20 25 30 Gly Lys Val Ile Thr Gly Gln Lys Ile LysLeu Arg 35 40 55 45 PRT Haemophilus influenzae SITE (1)..(45) AcmA cellwall binding domain homologue 55 Tyr Thr Val Thr Glu Gly Asp Thr Leu LysAsp Val Leu Val Leu Ser 1 5 10 15 Gly Leu Asp Asp Ser Ser Val Gln ProLeu Ile Ala Leu Asp Pro Glu 20 25 30 Leu Ala His Leu Lys Ala Gly Gln GlnPhe Tyr Trp Ile 35 40 45 56 45 PRT Haemophilus influenzae SITE (1)..(45)AcmA cell wall binding domain homologue 56 Tyr Lys Val Asn Lys Gly AspThr Met Phe Leu Ile Ala Tyr Leu Ala 1 5 10 15 Gly Ile Asp Val Lys GluLeu Ala Ala Leu Asn Asn Leu Ser Glu Pro 20 25 30 Asn Tyr Asn Leu Ser LeuGly Gln Val Leu Lys Ile Ser 35 40 45 57 44 PRT Haemophilus somnus SITE(1)..(44) AcmA cell wall binding domain homologue 57 Tyr Lys Val Arg LysGly Asp Thr Met Phe Leu Ile Ala Tyr Ile Ser 1 5 10 15 Gly Met Asp IleLys Glu Leu Ala Thr Leu Asn Asn Met Ser Glu Pro 20 25 30 Tyr His Leu SerIle Gly Gln Val Leu Lys Ile Ala 35 40 58 43 PRT Helicobacter pylori SITE(1)..(43) AcmA cell wall binding domain homologue 58 His Val Val Leu ProLys Glu Thr Leu Ser Ser Ile Ala Lys Arg Tyr 1 5 10 15 Gln Val Ser IleSer Asn Ile Gln Leu Ala Asn Asp Leu Lys Asp Ser 20 25 30 Asn Ile Phe IleHis Gln Arg Leu Ile Ile Arg 35 40 59 44 PRT Pseudomonas aeruginosa SITE(1)..(44) AcmA cell wall binding domain homologue 59 Tyr Ile Val Arg ArgGly Asp Thr Leu Tyr Ser Ile Ala Phe Arg Phe 1 5 10 15 Gly Trp Asp TrpLys Ala Leu Ala Ala Arg Asn Gly Ile Ala Pro Pro 20 25 30 Tyr Thr Ile GlnVal Gly Gln Ala Ile Gln Phe Gly 35 40 60 44 PRT Pseudomonas putida SITE(1)..(44) AcmA cell wall binding domain homologue 60 Tyr Ile Val Lys ProGly Asp Thr Leu Phe Ser Ile Ala Phe Arg Tyr 1 5 10 15 Gly Trp Asp TyrLys Glu Leu Ala Ala Arg Asn Gly Ile Pro Ala Pro 20 25 30 Tyr Thr Ile ArgPro Gly Gln Pro Ile Arg Phe Ser 35 40 61 44 PRT Sinorhizobium melilotiSITE (1)..(44) AcmA cell wall binding domain homologue 61 Ile Met ValArg Gln Gly Asp Thr Val Thr Val Leu Ala Arg Arg Phe 1 5 10 15 Gly ValPro Glu Lys Glu Ile Leu Lys Ala Asn Gly Leu Lys Ser Ala 20 25 30 Ser GlnVal Glu Pro Gly Gln Arg Leu Val Ile Pro 35 40 62 44 PRT Synechocystissp. SITE (1)..(44) AcmA cell wall binding domain homologue 62 His GlnVal Lys Glu Gly Glu Ser Leu Trp Gln Ile Ser Gln Ala Phe 1 5 10 15 GlnVal Asp Ala Lys Ala Ile Ala Leu Ala Asn Ser Ile Ser Thr Asp 20 25 30 ThrGlu Leu Gln Ala Gly Gln Val Leu Asn Ile Pro 35 40 63 44 PRTSynechocystis sp. SITE (1)..(44) AcmA cell wall binding domain homologue63 His Val Val Lys Ala Gly Glu Thr Ile Asp Ser Ile Ala Ala Gln Tyr 1 510 15 Gln Leu Val Pro Ala Thr Leu Ile Ser Val Asn Asn Gln Leu Ser Ser 2025 30 Gly Gln Val Thr Pro Gly Gln Thr Ile Leu Ile Pro 35 40 64 45 PRTAquifex aeolicus SITE (1)..(45) AcmA cell wall binding domain homologue64 Tyr Lys Val Lys Lys Gly Asp Ser Leu Trp Lys Ile Ala Lys Glu Tyr 1 510 15 Lys Thr Ser Ile Gly Lys Leu Leu Glu Leu Asn Pro Lys Leu Lys Asn 2025 30 Arg Lys Tyr Leu Arg Pro Gly Glu Lys Ile Cys Leu Lys 35 40 45 65 43PRT Aquifex aeolicus SITE (1)..(43) AcmA cell wall binding domainhomologue 65 Tyr Arg Val Lys Arg Gly Asp Ser Leu Ile Lys Ile Ala Lys LysPhe 1 5 10 15 Gly Val Ser Val Lys Glu Ile Lys Arg Val Asn Lys Leu LysGly Asn 20 25 30 Arg Ile Tyr Val Gly Gln Lys Leu Lys Ile Pro 35 40 66 43PRT Aquifex aeolicus SITE (1)..(43) AcmA cell wall binding domainhomologue 66 Tyr Arg Val Arg Arg Gly Asp Thr Leu Ile Lys Ile Ala Lys ArgPhe 1 5 10 15 Arg Thr Ser Val Lys Glu Ile Lys Arg Ile Asn Arg Leu LysGly Asn 20 25 30 Leu Ile Arg Val Gly Gln Lys Leu Lys Ile Pro 35 40 67 44PRT Volvox carteri SITE (1)..(44) AcmA cell wall binding domainhomologue 67 Tyr Thr Ile Gln Pro Gly Asp Thr Phe Trp Ala Ile Ala Gln ArgArg 1 5 10 15 Gly Thr Thr Val Asp Val Ile Gln Ser Leu Asn Pro Gly ValVal Pro 20 25 30 Thr Arg Leu Gln Val Gly Gln Val Ile Asn Val Pro 35 4068 44 PRT F. nagariensis SITE (1)..(44) AcmA cell wall binding domainhomologue 68 Tyr Thr Ile Gln Pro Gly Asp Thr Phe Trp Ala Ile Ala Gln ArgArg 1 5 10 15 Gly Thr Thr Val Asp Val Ile Gln Ser Leu Asn Pro Gly ValAsn Pro 20 25 30 Ala Arg Leu Gln Val Gly Gln Val Ile Asn Val Pro 35 4069 44 PRT Staphylococcus aureus SITE (1)..(44) AcmA cell wall bindingdomain homologue 69 His Val Val Lys Pro Gly Asp Thr Val Asn Asp Ile AlaLys Ala Asn 1 5 10 15 Gly Thr Thr Ala Asp Lys Ile Ala Ala Asp Asn LysLeu Ala Asp Lys 20 25 30 Asn Met Ile Lys Pro Gly Gln Glu Leu Val Val Asp35 40 70 44 PRT Staphylococcus aureus SITE (1)..(44) AcmA cell wallbinding domain homologue 70 Tyr Thr Val Lys Lys Gly Asp Thr Leu Ser AlaIle Ala Leu Lys Tyr 1 5 10 15 Lys Thr Thr Val Ser Asn Ile Gln Asn ThrAsn Asn Ile Ala Asn Pro 20 25 30 Asn Leu Ile Phe Ile Gly Gln Lys Leu LysVal Pro 35 40 71 44 PRT Colletotrichum SITE (1)..(44) AcmA cell wallbinding domain homologue 71 His Lys Val Lys Ser Gly Glu Ser Leu Thr ThrIle Ala Glu Lys Tyr 1 5 10 15 Asp Thr Gly Ile Cys Asn Ile Ala Lys LeuAsn Asn Leu Ala Asp Pro 20 25 30 Asn Phe Ile Asp Leu Asn Gln Asp Leu GlnIle Pro 35 40 72 44 PRT Colletotrichum lindemuthianum SITE (1)..(44)AcmA cell wall binding domain homologue 72 Tyr Ser Val Val Ser Gly AspThr Leu Thr Ser Ile Ala Gln Ala Leu 1 5 10 15 Gln Ile Thr Leu Gln SerLeu Lys Asp Ala Asn Pro Gly Val Val Pro 20 25 30 Glu His Leu Asn Val GlyGln Lys Leu Asn Val Pro 35 40 73 43 PRT Colletotrichum chlamydophilaSITE (1)..(43) AcmA cell wall binding domain homologue 73 Ile Val TyrArg Glu Gly Asp Ser Leu Ser Lys Ile Ala Lys Lys Tyr 1 5 10 15 Lys LeuSer Val Thr Glu Leu Lys Lys Ile Asn Lys Leu Asp Ser Asp 20 25 30 Ala IleTyr Ala Gly Gln Arg Leu Cys Leu Gln 35 40 74 43 PRT Colletotrichumpneumoniae SITE (1)..(43) AcmA cell wall binding domain homologue 74 TyrVal Val Gln Asp Gly Asp Ser Leu Trp Leu Ile Ala Lys Arg Phe 1 5 10 15Gly Ile Pro Met Asp Lys Ile Ile Gln Lys Asn Gly Leu Asn His His 20 25 30Arg Leu Phe Pro Gly Lys Val Leu Lys Leu Pro 35 40 75 43 PRTColletotrichum pneumoniae SITE (1)..(43) AcmA cell wall binding domainhomologue 75 Val Val Val Lys Lys Gly Asp Phe Leu Glu Arg Ile Ala Arg AlaAsn 1 5 10 15 His Thr Thr Val Ala Lys Leu Met Gln Ile Asn Asp Leu ThrThr Thr 20 25 30 Gln Leu Lys Ile Gly Gln Val Ile Lys Val Pro 35 40 76 46PRT Colletotrichum pneumoniae SITE (1)..(46) Acma cell wall bindingdomain homologue 76 Tyr Ile Val Gln Glu Gly Asp Ser Pro Trp Thr Ile AlaLeu Arg Asn 1 5 10 15 His Ile Arg Leu Asp Asp Leu Leu Lys Met Asn AspLeu Asp Glu Tyr 20 25 30 Lys Ala Arg Arg Leu Lys Pro Gly Asp Gln Leu ArgIle Arg 35 40 45 77 43 PRT Chlamydia trachomatis SITE (1)..(43) AcmAcell wall binding domain homologue 77 Val Ile Val Lys Lys Gly Asp PheLeu Glu Arg Ile Ala Arg Ser Asn 1 5 10 15 His Thr Thr Val Ser Ala LeuMet Gln Leu Asn Asp Leu Ser Ser Thr 20 25 30 Gln Leu Gln Ile Gly Gln ValLeu Arg Val Pro 35 40 78 46 PRT Chlamydia trachomatis SITE (1)..(46)AcmA cell wall binding domain homologue 78 Tyr Val Val Lys Glu Gly AspSer Pro Trp Ala Ile Ala Leu Ser Asn 1 5 10 15 Gly Ile Arg Leu Asp GluLeu Leu Lys Leu Asn Gly Leu Asp Glu Gln 20 25 30 Lys Ala Arg Arg Leu ArgPro Gly Asp Arg Leu Arg Ile Arg 35 40 45 79 43 PRT Chlamydia trachomatisSITE (1)..(43) AcmA cell wall binding domain homologue 79 His Ile ValLys Gln Gly Glu Thr Leu Ser Lys Ile Ala Ser Lys Tyr 1 5 10 15 Asn IlePro Val Val Glu Leu Lys Lys Leu Asn Lys Leu Asn Ser Asp 20 25 30 Thr IlePhe Thr Asp Gln Arg Ile Arg Leu Pro 35 40 80 43 PRT Prevotellaintermedia SITE (1)..(43) AcmA cell wall binding domain homologue 80 HisThr Val Arg Ser Asn Glu Ser Leu Tyr Asp Ile Ser Gln Gln Tyr 1 5 10 15Gly Val Arg Leu Lys Asn Ile Met Lys Ala Asn Arg Lys Ile Val Lys 20 25 30Arg Gly Ile Lys Ala Gly Asp Arg Val Val Leu 35 40 81 44 PRT Leuconostocoenos bacteriophage 10MC SITE (1)..(44) AcmA cell wall binding domainhomologue 81 Tyr Thr Val Gln Ser Gly Asp Thr Leu Gly Ala Ile Ala Ala LysTyr 1 5 10 15 Gly Thr Thr Tyr Gln Lys Leu Ala Ser Leu Asn Gly Ile GlySer Pro 20 25 30 Tyr Ile Ile Ile Pro Gly Glu Lys Leu Lys Val Ser 35 4082 44 PRT Leuconostoc oenos bacteriophage 10MC SITE (1)..(44) AcmA cellwall binding domain homologue 82 Tyr Lys Val Ala Ser Gly Asp Thr Leu SerAla Ile Ala Ser Lys Tyr 1 5 10 15 Gly Thr Ser Val Ser Lys Leu Val SerLeu Asn Gly Leu Lys Asn Ala 20 25 30 Asn Tyr Ile Tyr Val Gly Glu Asn LeuLys Ile Lys 35 40 83 44 PRT Oenococcus oeni SITE (1)..(44) AcmA cellwall binding domain homologue 83 Tyr Thr Val Arg Ser Gly Asp Thr Leu GlyAla Ile Ala Ala Lys Tyr 1 5 10 15 Gly Thr Thr Tyr Gln Lys Leu Ala SerLeu Asn Gly Ile Gly Ser Pro 20 25 30 Tyr Ile Ile Ile Pro Gly Glu Lys LeuLys Val Ser 35 40 84 44 PRT Oenococcus oeni SITE (1)..(44) AcmA cellwall binding domain homologue 84 Tyr Lys Val Ala Ser Gly Asp Thr Leu SerAla Ile Ala Ser Lys Tyr 1 5 10 15 Gly Thr Ser Val Ser Lys Leu Val SerLeu Asn Gly Leu Lys Asn Ala 20 25 30 Asn Tyr Ile Tyr Val Gly Gln Thr LeuArg Ile Lys 35 40 85 44 PRT Thermotoga maritima SITE (1)..(44) AcmA cellwall binding domain homologue 85 Tyr Lys Val Gln Lys Asn Asp Thr Leu TyrSer Ile Ser Leu Asn Phe 1 5 10 15 Gly Ile Ser Pro Ser Leu Leu Leu AspTrp Asn Pro Gly Leu Asp Pro 20 25 30 His Ser Leu Arg Val Gly Gln Glu IleVal Ile Pro 35 40 86 43 PRT Thermotoga maritima SITE (1)..(43) AcmA cellwall binding domain homologue 86 Tyr Thr Val Lys Lys Gly Asp Thr Leu AspAla Ile Ala Lys Arg Phe 1 5 10 15 Phe Thr Thr Ala Thr Phe Ile Lys GluAla Asn Gln Leu Lys Ser Tyr 20 25 30 Thr Ile Tyr Ala Gly Gln Lys Leu PheIle Pro 35 40 87 44 PRT Thermotoga maritima SITE (1)..(44) AcmA cellwall binding domain homologue 87 His Val Val Lys Arg Gly Glu Thr Leu TrpSer Ile Ala Asn Gln Tyr 1 5 10 15 Gly Val Arg Val Gly Asp Ile Val LeuIle Asn Arg Leu Glu Asp Pro 20 25 30 Asp Arg Ile Val Ala Gly Gln Val LeuLys Ile Gly 35 40 88 44 PRT Treponema pallidum SITE (1)..(44) AcmA cellwall binding domain homologue 88 His Thr Ile Arg Ser Gly Asp Thr Leu TyrAla Leu Ala Arg Arg Tyr 1 5 10 15 Gly Leu Gly Val Asp Thr Leu Lys AlaHis Asn Arg Ala His Ser Ala 20 25 30 Thr His Leu Lys Ile Gly Gln Lys LeuIle Ile Pro 35 40 89 44 PRT Treponema pallidum SITE (1)..(44) AcmA cellwall binding domain homologue 89 His Val Val Gln Gln Gly Asp Thr Leu TrpSer Leu Ala Lys Arg Tyr 1 5 10 15 Gly Val Ser Val Glu Asn Leu Ala GluGlu Asn Asn Leu Ala Val Asp 20 25 30 Ala Thr Leu Ser Leu Gly Met Ile LeuLys Thr Pro 35 40 90 44 PRT Treponema pallidum SITE (1)..(44) AcmA cellwall binding domain homologue 90 Tyr Glu Val Arg Glu Gly Asp Val Val GlyArg Ile Ala Gln Arg Tyr 1 5 10 15 Asp Ile Ser Gln Asp Ala Ile Ile SerLeu Asn Lys Leu Arg Ser Thr 20 25 30 Arg Ala Leu Gln Val Gly Gln Leu LeuLys Ile Pro 35 40 91 44 PRT Treponema pallidum SITE (1)..(44) AcmA cellwall binding domain homologue 91 His Val Ile Ala Lys Gly Glu Thr Leu PheSer Leu Ser Arg Arg Tyr 1 5 10 15 Gly Val Pro Leu Ser Ala Leu Ala GlnAla Asn Asn Leu Ala Asn Val 20 25 30 His Gln Leu Val Pro Gly Gln Arg IleVal Val Pro 35 40 92 44 PRT Borrelia burgdorferi SITE (1)..(44) AcmAcell wall binding domain homologue 92 His Lys Ile Lys Pro Gly Glu ThrLeu Ser His Val Ala Ala Arg Tyr 1 5 10 15 Gln Ile Thr Ser Glu Thr LeuIle Ser Phe Asn Glu Ile Lys Asp Val 20 25 30 Arg Asn Ile Lys Pro Asn SerVal Ile Lys Val Pro 35 40 93 43 PRT Borrelia burgdorferi SITE (1)..(43)AcmA cell wall binding domain homologue 93 Tyr Ile Val Lys Lys Asn AspSer Ile Ser Ser Ile Ala Ser Ala Tyr 1 5 10 15 Asn Val Pro Lys Val AspIle Leu Asp Ser Asn Asn Leu Asp Asn Glu 20 25 30 Val Leu Phe Leu Gly GlnLys Leu Phe Ile Pro 35 40 94 43 PRT Borrelia burgdorferi SITE (1)..(43)AcmA cell wall binding domain homologue 94 Tyr Lys Val Val Lys Gly AspThr Leu Phe Ser Ile Ala Ile Lys Tyr 1 5 10 15 Lys Val Lys Val Ser AspLeu Lys Arg Ile Asn Lys Leu Asn Val Asp 20 25 30 Asn Ile Lys Ala Gly GlnIle Leu Ile Ile Pro 35 40 95 43 PRT Borrelia burgdorferi SITE (1)..(43)AcmA cell wall binding domain homologue 95 Tyr Thr Ala Lys Glu Gly AspThr Ile Glu Ser Ile Ser Lys Leu Val 1 5 10 15 Gly Leu Ser Gln Glu GluIle Ile Ala Trp Asn Asp Leu Arg Ser Lys 20 25 30 Asp Leu Lys Val Gly MetLys Leu Val Leu Thr 35 40 96 43 PRT Borrelia burgdorferi SITE (1)..(43)AcmA cell wall binding domain homologue 96 Tyr Met Val Arg Lys Gly AspSer Leu Ser Lys Leu Ser Gln Asp Phe 1 5 10 15 Asp Ile Ser Ser Lys AspIle Leu Lys Phe Asn Phe Leu Asn Asp Asp 20 25 30 Lys Leu Lys Ile Gly GlnGln Leu Phe Leu Lys 35 40 97 43 PRT Borrelia burgdorferi SITE (1)..(43)AcmA cell wall binding domain homologue 97 His Tyr Val Lys Arg Gly GluThr Leu Gly Arg Ile Ala Tyr Ile Tyr 1 5 10 15 Gly Val Thr Ala Lys AspLeu Val Ala Leu Asn Gly Asn Arg Ala Ile 20 25 30 Asn Leu Lys Ala Gly SerLeu Leu Asn Val Leu 35 40 98 43 PRT Borrelia burgdorferi SITE (1)..(43)AcmA cell wall binding domain homologue 98 His Ser Val Ala Val Gly GluThr Leu Tyr Ser Ile Ala Arg His Tyr 1 5 10 15 Gly Val Leu Ile Glu AspLeu Lys Asn Trp Asn Asn Leu Ser Ser Asn 20 25 30 Asn Ile Met His Asp GlnLys Leu Lys Ile Phe 35 40 99 43 PRT Borrelia burgdorferi SITE (1)..(43)AcmA cell wall binding domain homologue 99 Tyr Lys Val Lys Lys Gly AspThr Phe Phe Lys Ile Ala Asn Lys Ile 1 5 10 15 Asn Gly Trp Gln Ser GlyIle Ala Thr Ile Asn Leu Leu Asp Ser Pro 20 25 30 Ala Val Ser Val Gly GlnGlu Ile Leu Ile Pro 35 40 100 44 PRT Lactobacillus SITE (1)..(44) AcmAcell wall binding domain homologue 100 Tyr Thr Val Val Ser Gly Asp SerTrp Trp Lys Ile Ala Gln Arg Asn 1 5 10 15 Gly Leu Ser Met Tyr Thr LeuAla Ser Gln Asn Gly Lys Ser Ile Tyr 20 25 30 Ser Thr Ile Tyr Pro Gly AsnLys Leu Ile Ile Lys 35 40 101 43 PRT Bacillus subtilis SITE (1)..(43)AcmA cell wall binding domain homologue 101 Ile Lys Val Lys Lys Gly AspThr Leu Trp Asp Leu Ser Arg Lys Tyr 1 5 10 15 Asp Thr Thr Ile Ser LysIle Lys Ser Glu Asn His Leu Arg Ser Asp 20 25 30 Ile Ile Tyr Val Gly GlnThr Leu Ser Ile Asn 35 40 102 43 PRT Bacillus subtilis SITE (1)..(43)AcmA cell wall binding domain homologue 102 Tyr Lys Val Lys Ser Gly AspSer Leu Trp Lys Ile Ser Lys Lys Tyr 1 5 10 15 Gly Met Thr Ile Asn GluLeu Lys Lys Leu Asn Gly Leu Lys Ser Asp 20 25 30 Leu Leu Arg Val Gly GlnVal Leu Lys Leu Lys 35 40 103 43 PRT Bacillus subtilis SITE (1)..(43)AcmA cell wall binding domain homologue 103 Tyr Lys Val Lys Ser Gly AspSer Leu Ser Lys Ile Ala Ser Lys Tyr 1 5 10 15 Gly Thr Thr Val Ser LysLeu Lys Ser Leu Asn Gly Leu Lys Ser Asp 20 25 30 Val Ile Tyr Val Asn GlnVal Leu Lys Val Lys 35 40 104 44 PRT Bacillus subtilis SITE (1)..(44)AcmA cell wall binding domain homologue 104 Cys Ile Val Gln Gln Glu AspThr Ile Glu Arg Leu Cys Glu Arg Tyr 1 5 10 15 Glu Ile Thr Ser Gln GlnLeu Ile Arg Met Asn Ser Leu Ala Leu Asp 20 25 30 Asp Glu Leu Lys Ala GlyGln Ile Leu Tyr Ile Pro 35 40 105 43 PRT Bacillus subtilis SITE(1)..(43) AcmA cell wall binding domain homologue 105 Met Val Lys GlnGly Asp Thr Leu Ser Ala Ile Ala Ser Gln Tyr Arg 1 5 10 15 Thr Thr ThrAsn Asp Ile Thr Glu Thr Asn Glu Ile Pro Asn Pro Asp 20 25 30 Ser Leu ValVal Gly Gln Thr Ile Val Ile Pro 35 40 106 44 PRT Bacillus subtilis SITE(1)..(44) AcmA cell wall binding domain homologue 106 Tyr Asp Val LysArg Gly Asp Thr Leu Thr Ser Ile Ala Arg Gln Phe 1 5 10 15 Asn Thr ThrAla Ala Glu Leu Ala Arg Val Asn Arg Ile Gln Leu Asn 20 25 30 Thr Val LeuGln Ile Gly Phe Arg Leu Tyr Ile Pro 35 40 107 43 PRT Bacillus subtilisSITE (1)..(43) AcmA cell wall binding domain homologue 107 Ile Lys ValLys Ser Gly Asp Ser Leu Trp Lys Leu Ala Gln Thr Tyr 1 5 10 15 Asn ThrSer Val Ala Ala Leu Thr Ser Ala Asn His Leu Ser Thr Thr 20 25 30 Val LeuSer Ile Gly Gln Thr Leu Thr Ile Pro 35 40 108 43 PRT Bacillus subtilisSITE (1)..(43) AcmA cell wall binding domain homologue 108 Tyr Thr ValLys Ser Gly Asp Ser Leu Trp Leu Ile Ala Asn Glu Phe 1 5 10 15 Lys MetThr Val Gln Glu Leu Lys Lys Leu Asn Gly Leu Ser Ser Asp 20 25 30 Leu IleArg Ala Gly Gln Lys Leu Lys Val Ser 35 40 109 43 PRT Bacillus subtilisSITE (1)..(43) AcmA cell wall binding domain homologue 109 Tyr Lys ValGln Leu Gly Asp Ser Leu Trp Lys Ile Ala Asn Lys Val 1 5 10 15 Asn MetSer Ile Ala Glu Leu Lys Val Leu Asn Asn Leu Lys Ser Asp 20 25 30 Thr IleTyr Val Asn Gln Val Leu Lys Thr Lys 35 40 110 43 PRT Bacillus subtilisSITE (1)..(43) AcmA cell wall binding domain homologue 110 Tyr Thr ValLys Ser Gly Asp Ser Leu Trp Lys Ile Ala Asn Asn Tyr 1 5 10 15 Asn LeuThr Val Gln Gln Ile Arg Asn Ile Asn Asn Leu Lys Ser Asp 20 25 30 Val LeuTyr Val Gly Gln Val Leu Lys Leu Thr 35 40 111 43 PRT Bacillus subtilisSITE (1)..(43) AcmA cell wall binding domain homologue 111 Tyr Thr ValLys Ser Gly Asp Ser Leu Trp Val Ile Ala Gln Lys Phe 1 5 10 15 Asn ValThr Ala Gln Gln Ile Arg Glu Lys Asn Asn Leu Lys Thr Asp 20 25 30 Val LeuGly Val Gly Gln Lys Leu Val Ile Ser 35 40 112 43 PRT Bacillus subtilisSITE (1)..(43) AcmA cell wall binding domain homologue 112 Ile Lys ValLys Ser Gly Asp Ser Leu Trp Lys Leu Ser Arg Gln Tyr 1 5 10 15 Asp ThrThr Ile Ser Ala Leu Lys Ser Glu Asn Lys Leu Lys Ser Thr 20 25 30 Val LeuTyr Val Gly Gln Ser Leu Lys Val Pro 35 40 113 43 PRT Bacillus subtilisSITE (1)..(43) AcmA cell wall binding domain homologue 113 Tyr Thr ValAla Tyr Gly Asp Ser Leu Trp Met Ile Ala Lys Asn His 1 5 10 15 Lys MetSer Val Ser Glu Leu Lys Ser Leu Asn Ser Leu Ser Ser Asp 20 25 30 Leu IleArg Pro Gly Gln Lys Leu Lys Ile Lys 35 40 114 43 PRT Bacillus subtilisSITE (1)..(43) AcmA cell wall binding domain homologue 114 Tyr Thr ValLys Leu Gly Asp Ser Leu Trp Lys Ile Ala Asn Ser Leu 1 5 10 15 Asn MetThr Val Ala Glu Leu Lys Thr Leu Asn Gly Leu Thr Ser Asp 20 25 30 Thr LeuTyr Pro Lys Gln Val Leu Lys Ile Gly 35 40 115 43 PRT Bacillus subtilisSITE (1)..(43) AcmA cell wall binding domain homologue 115 Tyr Lys ValLys Ala Gly Asp Ser Leu Trp Lys Ile Ala Asn Arg Leu 1 5 10 15 Gly ValThr Val Gln Ser Ile Arg Asp Lys Asn Asn Leu Ser Ser Asp 20 25 30 Val LeuGln Ile Gly Gln Val Leu Thr Ile Ser 35 40 116 43 PRT Bacillus subtilisSITE (1)..(43) AcmA cell wall binding domain homologue 116 Ile Thr ValGln Lys Gly Asp Thr Leu Trp Gly Ile Ser Gln Lys Asn 1 5 10 15 Gly ValAsn Leu Lys Asp Leu Lys Glu Trp Asn Lys Leu Thr Ser Asp 20 25 30 Lys IleIle Ala Gly Glu Lys Leu Thr Ile Ser 35 40 117 43 PRT Bacillus subtilisSITE (1)..(43) AcmA cell wall binding domain homologue 117 Tyr Thr IleLys Ala Gly Asp Thr Leu Ser Lys Ile Ala Gln Lys Phe 1 5 10 15 Gly ThrThr Val Asn Asn Leu Lys Val Trp Asn Asn Leu Ser Ser Asp 20 25 30 Met IleTyr Ala Gly Ser Thr Leu Ser Val Lys 35 40 118 43 PRT Bacillus subtilisSITE (1)..(43) AcmA cell wall binding domain homologue 118 His His ValThr Pro Gly Glu Thr Leu Ser Ile Ile Ala Ser Lys Tyr 1 5 10 15 Asn ValSer Leu Gln Gln Leu Met Glu Leu Asn His Phe Lys Ser Asp 20 25 30 Gln IleTyr Ala Gly Gln Ile Ile Lys Ile Arg 35 40 119 44 PRT Bacillus subtilisSITE (1)..(44) AcmA cell wall binding domain homologue 119 Tyr His ValLys Lys Gly Asp Thr Leu Ser Gly Ile Ala Ala Ser His 1 5 10 15 Gly AlaSer Val Lys Thr Leu Gln Ser Ile Asn His Ile Thr Asp Pro 20 25 30 Asn HisIle Lys Ile Gly Gln Val Ile Lys Leu Pro 35 40 120 45 PRT Bacillussubtilis SITE (1)..(45) AcmA cell wall binding domain homologue 120 HisIle Val Gln Lys Gly Asp Ser Leu Trp Lys Ile Ala Glu Lys Tyr 1 5 10 15Gly Val Asp Val Glu Glu Val Lys Lys Leu Asn Thr Gln Leu Ser Asn 20 25 30Pro Asp Leu Ile Met Pro Gly Met Lys Ile Lys Val Pro 35 40 45 121 43 PRTBacillus subtilis SITE (1)..(43) AcmA cell wall binding domain homologue121 His Ile Val Gly Pro Gly Asp Ser Leu Phe Ser Ile Gly Arg Arg Tyr 1 510 15 Gly Ala Ser Val Asp Gln Ile Arg Gly Val Asn Gly Leu Asp Glu Thr 2025 30 Asn Ile Val Pro Gly Gln Ala Leu Leu Ile Pro 35 40 122 43 PRTBacillus subtilis SITE (1)..(43) AcmA cell wall binding domain homologue122 Tyr Gln Val Lys Gln Gly Asp Thr Leu Asn Ser Ile Ala Ala Asp Phe 1 510 15 Arg Ile Ser Thr Ala Ala Leu Leu Gln Ala Asn Pro Ser Leu Gln Ala 2025 30 Gly Leu Thr Ala Gly Gln Ser Ile Val Ile Pro 35 40 123 44 PRTBacillus subtilis phage PBSX SITE (1)..(44) AcmA cell wall bindingdomain homologue 123 Tyr Val Val Lys Gln Gly Asp Thr Leu Thr Ser Ile AlaArg Ala Phe 1 5 10 15 Gly Val Thr Val Ala Gln Leu Gln Glu Trp Asn AsnIle Glu Asp Pro 20 25 30 Asn Leu Ile Arg Val Gly Gln Val Leu Ile Val Ser35 40 124 45 PRT Bacillus subtilis phage PZA SITE (1)..(45) AcmA cellwall binding domain homologue 124 Tyr Lys Val Lys Ser Gly Asp Asn LeuThr Lys Ile Ala Lys Lys His 1 5 10 15 Asn Thr Thr Val Ala Thr Leu LeuLys Leu Asn Pro Ser Ile Lys Asp 20 25 30 Pro Asn Met Ile Arg Val Gly GlnThr Ile Asn Val Thr 35 40 45 125 45 PRT Bacillus subtilis phage PZA SITE(1)..(45) AcmA cell wall binding domain homologue 125 His Lys Val LysSer Gly Asp Thr Leu Ser Lys Ile Ala Val Asp Asn 1 5 10 15 Lys Thr ThrVal Ser Arg Leu Met Ser Leu Asn Pro Glu Ile Thr Asn 20 25 30 Pro Asn HisIle Lys Val Gly Gln Thr Ile Arg Leu Ser 35 40 45 126 45 PRT Bacillussubtilis phage B103 SITE (1)..(45) AcmA cell wall binding domainhomologue 126 His Val Val Lys Lys Gly Asp Thr Leu Ser Glu Ile Ala LysLys Ile 1 5 10 15 Lys Thr Ser Thr Lys Thr Leu Leu Glu Leu Asn Pro ThrIle Lys Asn 20 25 30 Pro Asn Lys Ile Tyr Val Gly Gln Arg Ile Asn Val Gly35 40 45 127 45 PRT Bacillus subtilis phage B103 SITE (1)..(45) AcmAcell wall binding domain homologue 127 Tyr Lys Ile Lys Arg Gly Glu ThrLeu Thr Gly Ile Ala Lys Lys Asn 1 5 10 15 Lys Thr Thr Val Ser Gln LeuMet Lys Leu Asn Pro Asn Ile Lys Asn 20 25 30 Ala Asn Asn Ile Tyr Ala GlyGln Thr Ile Arg Leu Lys 35 40 45 128 44 PRT Bacillus sphaericus SITE(1)..(44) AcmA cell wall binding domain homologue 128 Ile Leu Ile ArgPro Gly Asp Ser Leu Trp Tyr Phe Ser Asp Leu Phe 1 5 10 15 Lys Ile ProLeu Gln Leu Leu Leu Asp Ser Asn Arg Asn Ile Asn Pro 20 25 30 Gln Leu LeuGln Val Gly Gln Arg Ile Gln Ile Pro 35 40 129 44 PRT Bacillus sphaericusSITE (1)..(44) AcmA cell wall binding domain homologue 129 Tyr Thr IleThr Gln Gly Asp Ser Leu Trp Gln Ile Ala Gln Asn Lys 1 5 10 15 Asn LeuPro Leu Asn Ala Ile Leu Leu Val Asn Pro Glu Ile Gln Pro 20 25 30 Ser ArgLeu His Ile Gly Gln Thr Ile Gln Val Pro 35 40 130 44 PRT Salmonelladublin SITE (1)..(44) AcmA cell wall binding domain homologue 130 TyrThr Val Lys Lys Gly Asp Thr Leu Phe Tyr Ile Ala Trp Ile Thr 1 5 10 15Gly Asn Asp Phe Arg Asp Leu Ala Gln Arg Asn Ser Ile Ser Ala Pro 20 25 30Tyr Ser Leu Asn Val Gly Gln Thr Leu Gln Val Gly 35 40 131 45 PRTEscherichia coli SITE (1)..(45) AcmA cell wall binding domain homologue131 Tyr Val Val Ser Thr Gly Asp Thr Leu Ser Ser Ile Leu Asn Gln Tyr 1 510 15 Gly Ile Asp Met Gly Asp Ile Ser Gln Leu Ala Ala Ala Asp Lys Glu 2025 30 Leu Arg Asn Leu Lys Ile Gly Gln Gln Leu Ser Trp Thr 35 40 45 13243 PRT Escherichia coli SITE (1)..(43) AcmA cell wall binding domainhomologue 132 Tyr Thr Val Arg Ser Gly Asp Thr Leu Ser Ser Ile Ala SerArg Leu 1 5 10 15 Gly Val Ser Thr Lys Asp Leu Gln Gln Trp Asn Lys LeuArg Gly Ser 20 25 30 Lys Leu Lys Pro Gly Gln Ser Leu Thr Ile Gly 35 40133 42 PRT Escherichia coli SITE (1)..(42) AcmA cell wall binding domainhomologue 133 Tyr Arg Val Arg Lys Gly Asp Ser Leu Ser Ser Ile Ala LysArg His 1 5 10 15 Gly Val Asn Ile Lys Asp Val Met Arg Trp Asn Ser AspThr Ala Asn 20 25 30 Leu Gln Pro Gly Asp Lys Leu Thr Leu Phe 35 40 13444 PRT Escherichia coli SITE (1)..(44) AcmA cell wall binding domainhomologue 134 Tyr Thr Val Lys Arg Gly Asp Thr Leu Tyr Arg Ile Ser ArgThr Thr 1 5 10 15 Gly Thr Ser Val Lys Glu Leu Ala Arg Leu Asn Gly IleSer Pro Pro 20 25 30 Tyr Thr Ile Glu Val Gly Gln Lys Leu Lys Leu Gly 3540 135 44 PRT Staphylococcus SITE (1)..(44) AcmA cell wall bindingdomain homologue 135 Tyr Thr Val Lys Lys Gly Asp Thr Leu Phe Tyr Ile AlaTrp Ile Thr 1 5 10 15 Gly Asn Asp Phe Arg Asp Leu Ala Gln Arg Asn AsnIle Gln Ala Pro 20 25 30 Tyr Ala Leu Asn Val Gly Gln Thr Leu Gln Val Gly35 40 136 43 PRT Drosophila melanogaster SITE (1)..(43) AcmA cell wallbinding domain homologue 136 Tyr Thr Val Gly Asn Arg Asp Thr Leu Thr SerVal Ala Ala Arg Phe 1 5 10 15 Asp Thr Thr Pro Ser Glu Leu Thr His LeuAsn Arg Leu Asn Ser Ser 20 25 30 Phe Ile Tyr Pro Gly Gln Gln Leu Leu ValPro 35 40 137 44 PRT Caenorhabditis elegans SITE (1)..(44) AcmA cellwall binding domain homologue 137 Arg Lys Val Lys Asn Gly Asp Thr LeuAsn Lys Leu Ala Ile Lys Tyr 1 5 10 15 Gln Val Asn Val Ala Glu Ile LysArg Val Asn Asn Met Val Ser Glu 20 25 30 Gln Asp Phe Met Ala Leu Ser LysVal Lys Ile Pro 35 40 138 43 PRT Caenorhabditis elegans SITE (1)..(43)AcmA cell wall binding domain homologue 138 Tyr Thr Ile Thr Glu Thr AspThr Leu Glu Arg Val Ala Ala Ser His 1 5 10 15 Asp Cys Thr Val Gly GluLeu Met Lys Leu Asn Lys Met Ala Ser Arg 20 25 30 Met Val Phe Pro Gly GlnLys Ile Leu Val Pro 35 40 139 44 PRT Caenorhabditis elegans SITE(1)..(44) AcmA cell wall binding domain homologue 139 Thr Glu Ile LysSer Gly Asp Ser Cys Trp Asn Ile Ala Ser Asn Ala 1 5 10 15 Lys Ile SerVal Glu Arg Leu Gln Gln Leu Asn Lys Gly Met Lys Cys 20 25 30 Asp Lys LeuPro Leu Gly Asp Lys Leu Cys Leu Ala 35 40 140 44 PRT Caenorhabditiselegans SITE (1)..(44) AcmA cell wall binding domain homologue 140 LeuLys Leu Lys Ala Glu Asp Thr Cys Phe Lys Ile Trp Ser Ser Gln 1 5 10 15Lys Leu Ser Glu Arg Gln Phe Leu Gly Met Asn Glu Gly Met Asp Cys 20 25 30Asp Lys Leu Lys Val Gly Lys Glu Val Cys Val Ala 35 40 141 44 PRTCaenorhabditis elegans SITE (1)..(44) AcmA cell wall binding domainhomologue 141 His Lys Ile Gln Lys Gly Asp Thr Cys Phe Lys Ile Trp ThrThr Asn 1 5 10 15 Lys Ile Ser Glu Lys Gln Phe Arg Asn Leu Asn Lys GlyLeu Asp Cys 20 25 30 Asp Lys Leu Glu Ile Gly Lys Glu Val Cys Ile Ser 3540 142 44 PRT Caenorhabditis elegans SITE (1)..(44) AcmA cell wallbinding domain homologue 142 Leu Lys Ile Lys Glu Gly Asp Thr Cys Tyr AsnIle Trp Thr Ser Gln 1 5 10 15 Lys Ile Ser Glu Gln Glu Phe Met Glu LeuAsn Lys Gly Leu Asp Cys 20 25 30 Asp Lys Leu Glu Ile Gly Lys Glu Val CysVal Thr 35 40 143 44 PRT Caenorhabditis elegans SITE (1)..(44) AcmA cellwall binding domain homologue 143 Tyr Arg Phe Lys Lys Gly Asp Thr CysTyr Lys Ile Trp Thr Ser His 1 5 10 15 Lys Met Ser Glu Lys Gln Phe ArgAla Leu Asn Arg Gly Ile Asp Cys 20 25 30 Asp Arg Leu Val Pro Gly Lys GluLeu Cys Val Gly 35 40 144 44 PRT Caenorhabditis elegans SITE (1)..(44)AcmA cell wall binding domain homologue 144 Ile Thr Val Lys Pro Gly AspThr Cys Phe Ser Ile Trp Thr Ser Gln 1 5 10 15 Lys Met Thr Gln Gln GlnPhe Met Asp Ile Asn Pro Glu Leu Asp Cys 20 25 30 Asp Lys Leu Glu Ile GlyLys Glu Val Cys Val Thr 35 40 145 44 PRT Caenorhabditis elegans SITE(1)..(44) AcmA cell wall binding domain homologue 145 Val Lys Ile AsnPro Gly Asp Thr Cys Phe Asn Ile Trp Thr Ser Gln 1 5 10 15 Arg Met ThrGln Gln Gln Phe Met Asp Leu Asn Lys Arg Leu Asp Cys 20 25 30 Asp Lys LeuGlu Val Gly Lys Glu Val Cys Val Thr 35 40 146 44 PRT Caenorhabditiselegans SITE (1)..(44) AcmA cell wall binding domain homologue 146 ValGln Ile Asn Pro Gly Asp Thr Cys Phe Lys Ile Trp Ser Ala Gln 1 5 10 15Lys Leu Thr Glu Gln Gln Phe Met Glu Leu Asn Lys Gly Leu Asp Cys 20 25 30Asp Arg Leu Glu Val Gly Lys Glu Val Cys Ile Ala 35 40 147 44 PRTCaenorhabditis elegans SITE (1)..(44) AcmA cell wall binding domainhomologue 147 Thr Glu Val Lys Glu Gly Asp Thr Cys Phe Lys Ile Trp SerAla His 1 5 10 15 Lys Ile Thr Glu Gln Gln Phe Met Glu Met Asn Arg GlyLeu Asp Cys 20 25 30 Asn Arg Leu Glu Val Gly Lys Glu Val Cys Ile Val 3540 148 44 PRT Caenorhabditis elegans SITE (1)..(44) AcmA cell wallbinding domain homologue 148 Ile Lys Val Lys Glu Gly Asp Thr Cys Phe LysIle Trp Ser Ala Gln 1 5 10 15 Lys Met Thr Glu Gln Gln Phe Met Glu MetAsn Arg Gly Leu Asp Cys 20 25 30 Asn Lys Leu Met Val Gly Lys Glu Val CysVal Ser 35 40 149 41 PRT Caenorhabditis elegans SITE (1)..(41) AcmA cellwall binding domain homologue 149 Ala Thr Ile Thr Pro Gly Asn Thr CysPhe Asn Ile Ser Val Ala Tyr 1 5 10 15 Gly Ile Asn Leu Thr Asp Leu GlnLys Thr Tyr Asp Cys Lys Ala Leu 20 25 30 Glu Val Gly Asp Thr Ile Cys ValSer 35 40 150 44 PRT Caenorhabditis elegans SITE (1)..(44) AcmA cellwall binding domain homologue 150 Ile Glu Val Ile Lys Gly Asp Thr CysTrp Phe Leu Glu Asn Ala Phe 1 5 10 15 Lys Thr Asn Gln Thr Glu Met GluArg Ala Asn Glu Gly Val Lys Cys 20 25 30 Asp Asn Leu Pro Ile Gly Arg MetMet Cys Val Trp 35 40 151 44 PRT Caenorhabditis elegans SITE (1)..(44)AcmA cell wall binding domain homologue 151 His Thr Ile Lys Ser Gly AspThr Cys Trp Lys Ile Ala Ser Glu Ala 1 5 10 15 Ser Ile Ser Val Gln GluLeu Glu Gly Leu Asn Ser Lys Lys Ser Cys 20 25 30 Ala Asn Leu Ala Val GlyLeu Ser Glu Gln Glu Phe 35 40 152 44 PRT Caenorhabditis elegans SITE(1)..(44) AcmA cell wall binding domain homologue 152 Ile His Val LysGlu Gly Asp Thr Cys Tyr Thr Ile Trp Thr Ser Gln 1 5 10 15 His Leu ThrGlu Lys Gln Phe Met Asp Met Asn Glu Glu Leu Asn Cys 20 25 30 Gly Met LeuGlu Ile Gly Asn Glu Val Cys Val Asp 35 40 153 41 PRT Caenorhabditiselegans SITE (1)..(41) AcmA cell wall binding domain homologue 153 AlaThr Val Thr Pro Gly Ser Ser Cys Tyr Thr Ile Ser Ala Ser Tyr 1 5 10 15Gly Leu Asn Leu Ala Glu Leu Gln Thr Thr Tyr Asn Cys Asp Ala Leu 20 25 30Gln Val Asp Asp Thr Ile Cys Val Ser 35 40 154 44 PRT Caenorhabditiselegans SITE (1)..(44) AcmA cell wall binding domain homologue 154 IleGlu Ile Leu Asn Gly Asp Thr Cys Gly Phe Leu Glu Asn Ala Phe 1 5 10 15Gln Thr Asn Asn Thr Glu Met Glu Ile Ala Asn Glu Gly Val Lys Cys 20 25 30Asp Asn Leu Pro Ile Gly Arg Met Met Cys Val Trp 35 40 155 46 PRTBacillus subtilis SITE (1)..(46) AcmA cell wall binding domain homologue155 His Thr Val Gln Lys Lys Glu Thr Leu Tyr Arg Ile Ser Met Lys Tyr 1 510 15 Tyr Lys Ser Arg Thr Gly Glu Glu Lys Ile Arg Ala Tyr Asn His Leu 2025 30 Asn Gly Asn Asp Val Tyr Thr Gly Gln Val Leu Asp Ile Pro 35 40 45156 49 PRT Citrobacter freundii SITE (1)..(49) AcmA cell wall bindingdomain homologue 156 Tyr Thr Leu Lys Thr Gly Glu Ser Val Ala Gln Leu SerLys Ser Gln 1 5 10 15 Gly Ile Ser Val Pro Val Ile Trp Ser Leu Asn LysHis Leu Tyr Ser 20 25 30 Ser Glu Ser Glu Met Met Lys Ala Ser Pro Gly GlnGln Ile Ile Leu 35 40 45 Pro 157 49 PRT Escherichia coli SITE (1)..(49)AcmA cell wall binding domain homologue 157 Tyr Thr Leu Lys Thr Gly GluThr Val Ala Asp Leu Ser Lys Ser Gln 1 5 10 15 Asp Ile Asn Leu Ser ThrIle Trp Ser Leu Asn Lys His Leu Tyr Ser 20 25 30 Ser Glu Ser Glu Met MetLys Ala Ala Pro Gly Gln Gln Ile Ile Leu 35 40 45 Pro 158 47 PRTMicrococcus luteus SITE (1)..(47) AcmA cell wall binding domainhomologue 158 Ile Val Val Lys Ser Gly Asp Ser Leu Trp Thr Leu Ala AsnGlu Tyr 1 5 10 15 Glu Val Glu Gly Gly Trp Thr Ala Leu Tyr Glu Ala AsnLys Gly Ala 20 25 30 Val Ser Asp Ala Ala Val Ile Tyr Val Gly Gln Glu LeuVal Leu 35 40 45 159 51 PRT Bacillus subtilis SITE (1)..(51) AcmA cellwall binding domain homologue 159 Ile Glu Val Gln Gln Gly Asp Thr LeuTrp Ser Ile Ala Asp Gln Val 1 5 10 15 Ala Asp Thr Lys Lys Ile Asn LysAsn Asp Phe Ile Glu Trp Val Ala 20 25 30 Asp Lys Asn Gln Leu Gln Thr SerAsp Ile Gln Pro Gly Asp Glu Leu 35 40 45 Val Ile Pro 50 160 55 PRTStreptococcus pyogenes SITE (1)..(55) AcmA cell wall binding domainhomologue 160 Tyr Thr Val Lys Tyr Gly Asp Thr Leu Ser Thr Ile Ala GluAla Met 1 5 10 15 Gly Ile Asp Val His Val Leu Gly Asp Ile Asn His IleAla Asn Ile 20 25 30 Asp Leu Ile Phe Pro Asp Thr Ile Leu Thr Ala Asn TyrAsn Gln His 35 40 45 Gly Gln Ala Thr Thr Leu Thr 50 55 161 57 PRTBacillus subtilis SITE (1)..(57) AcmA cell wall binding domain homologue161 Tyr Thr Val Lys Lys Gly Asp Thr Leu Trp Asp Ile Ala Gly Arg Phe 1 510 15 Tyr Gly Asn Ser Thr Gln Trp Arg Lys Ile Trp Asn Ala Asn Lys Thr 2025 30 Ala Met Ile Lys Arg Ser Lys Arg Asn Ile Arg Gln Pro Gly His Trp 3540 45 Ile Phe Pro Gly Gln Lys Leu Ile Pro 50 55 162 58 PRT Bacillussubtilis SITE (1)..(58) AcmA cell wall binding domain homologue 162 TyrThr Val Lys Lys Gly Asp Thr Leu Trp Asp Leu Ala Gly Lys Phe 1 5 10 15Tyr Gly Asp Ser Thr Lys Trp Arg Lys Ile Trp Lys Val Asn Lys Lys 20 25 30Ala Met Ile Lys Arg Ser Lys Arg Asn Ile Arg Gln Pro Gly His Trp 35 40 45Ile Phe Pro Gly Gln Lys Leu Lys Ile Pro 50 55 163 45 PRT Lactococcuslactis SITE (1)..(45) Consensus repeat, Xaa stands for any amino acid163 Tyr Xaa Val Lys Xaa Gly Asp Thr Leu Xaa Xaa Ile Ala Xaa Xaa Xaa 1 510 15 Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Asn Xaa Xaa Leu Xaa Xaa 2025 30 Xaa Xaa Xaa Ile Xaa Xaa Gly Gln Xaa Ile Xaa Val Xaa 35 40 45 16445 PRT Lactococcus lactis SITE (1)..(45) Consensus repeat, Xaa standsfor any amino acid 164 His Xaa Ile Arg Xaa Xaa Glu Ser Val Xaa Xaa LeuSer Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Ile Xaa Xaa Xaa XaaXaa Xaa Ile Xaa Xaa 20 25 30 Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Leu XaaIle Xaa 35 40 45 165 45 PRT Lactococcus lactis SITE (1)..(45) Consensusrepeat, Xaa stands for any amino acid 165 Xaa Xaa Leu Xaa Xaa Xaa XaaXaa Ile Xaa Xaa Val Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Val Xaa XaaXaa Xaa Xaa Val Xaa Leu Xaa 35 40 45

What is claimed is:
 1. A method for binding a proteinaceous substance tocell-wall material of a Gram-positive bacterium, said method comprising:treating said cell-wall material with a solution capable of removing acell-wall component selected from the group consisting of a protein, alipoteichoic acid, a carbohydrate, and mixtures thereof from saidcell-wall material; contacting said proteinaceous substance with saidcell-wall material; and providing said proteinaceous substance with animmunogenic determinant; wherein said proteinaceous substance comprisesan AcmA cell-wall binding domain, homolog, or functional derivativethereof.
 2. The method according to claim 1, wherein said solutioncomprises an acid selected from the group consisting of acetic acid(HAc), hydrochloric acid (HCl), sulphuric acid (H₂SO₄), trichloric acid(TCA), trifluoric acid (TFA), monochloric acid (MCA), and mixturesthereof.
 3. The method according to claim 1, wherein said Gram-positivebacterium is selected from the group consisting of a Lactococcus, aLactobacillus, a Bacillus and a Mycobacterium spp.
 4. The methodaccording to claim 1, wherein said immunogenic determinant is of apathogen origin.
 5. The method according to claim 4, wherein saidpathogen is Plasmodium falciparum.
 6. The method according to claim 5,wherein said proteinaceous substance is the malaria antigen MSA2 fusedto the lactococcal AcmA protein anchor.
 7. The method according to claim4, wherein said pathogen is Streptococcus pneumoniae.
 8. The methodaccording to claim 7, wherein said proteinaceous substance is theStreptococcal PpmA antigen fused to the lactococcal AcmA protein anchor.9. A method for obtaining cell-wall material from a Gram-positivebacterium, said method comprising: treating said cell-wall material witha solution capable of removing a cell-wall component from said cell-wallmaterial, said cell-wall component selected from the group consisting ofa protein, a lipoteichoic acid, a carbohydrate, and mixtures thereof,wherein said cell-wall material has an improved capacity for binding aproteinaceous substance comprising an AcmA cell wall binding domain,homolog or functional derivative thereof.
 10. The method according toclaim 9, wherein said proteinaceous substance further comprises areactive group.
 11. The method according to claim 10, wherein saidreactive group is selected from the group consisting of an antigenicdeterminant, an enzyme, an antibody, an antibiotic, a hormone, anaromatic substance, an inorganic particle, and a reporter molecule. 12.The-method according to claim 9 or 10, wherein said solution comprisesan acid selected from the group consisting of acetic acid (HAc),hydrochloric acid (HCl), sulphuric acid (H2SO4), trichloric acid (TCA),trifluoric acid (TEA), monochloric acid (MCA), and mixtures thereof. 13.The method according to claim 12, wherein said solution comprises 0.06to 1.2 M TCA.
 14. The method according to any one of claims 9-13,further comprising heating said cell-wall material in said solution. 15.The method according to any one of claims 9-14, further comprisingpelleting said cell-wall material from said solution.
 16. The methodaccording to any one of claims 9-15, wherein said cell-wall materialcomprises spherical peptidoglycan microparticles.
 17. The methodaccording to claim 16, wherein said Gram-positive bacterium is selectedfrom the group consisting of a Lactococcus, a Lactobacillus, a Bacillusand a Mycobacterium spp.
 18. The cell-wall material produced by themethod according to any one of claims 9-17.
 19. The cell-wall materialof claim 18, further comprising said proteinaceous substance.
 20. Thecell-wall material of claim 19, wherein said proteinaceous substancefurther comprises a reactive group.
 21. A pharmaceutical composition,comprising the cell-wall material of any one of claims 18-20.
 22. Thepharmaceutical composition of claim 21, wherein said pharmaceuticalcomposition is a vaccine.
 23. A method for immunizing a subject,comprising: administering the vaccine of claim 22 to a mucosal membraneof the subject.
 24. A method for generating a biocatalyst, comprising:preparing a biocatalyst comprising the cell-wall material of any one ofclaims 18-20.
 25. A product obtained by a process, said processcomprising: treating cell-wall material of a Gram-positive bacteriumwith a solution capable of removing a cell-wall component selected fromthe group consisting of a protein, a lipoteichoic acid, a carbohydrate,and mixtures thereof from said cell-wall material; contacting aproteinaceous substance with said cell-wall material; and providing saidproteinaceous substance with an immunogenic determinant.
 26. The productof the process according to claim 25, wherein said product comprisesbacterial ghosts.
 27. The product of the process according to claim 26,wherein said bacterial ghosts comprise spherical microparticles.
 28. Theproduct of the process according to claim 25, wherein said proteinaceoussubstance further comprises an AcmA cell-wall binding domain, homolog,or functional derivative thereof.
 29. The product of the processaccording to claim 25, wherein said proteinaceous substance furthercomprises a reactive group.
 30. A pharmaceutical composition comprisingthe product produced by the process according to claim
 25. 31. Thepharmaceutical composition of claim 30, wherein said pharmaceuticalcomposition is a vaccine.
 32. A method for binding a proteinaceoussubstance to cell-wall material of a Gram-positive bacterium, saidproteinaceous substance comprising an AcmA cell wall binding domain,homolog, or functional derivative thereof, said method comprising:treating said cell-wall material with a solution capable of removing acell-wall component selected from the group consisting of a protein, alipoteichoic acid, a carbohydrate, and mixtures thereof from saidcell-wall-material; and contacting said proteinaceous substance withsaid cell-wall material.
 33. The method according to claim 32, whereinsaid proteinaceous substance is contacted with said cell-wall materialat a pH that is lower than the calculated pI value of said AcmA cellwall binding domain, homolog, or functional derivative thereof.
 34. Aproteinaceous substance, comprising: an AcmA cell-wall binding domain,homolog, or functional derivative thereof, wherein said cell-wallbinding domain is a hybrid of at least two different AcmA cell wallbinding domains, homologs, or functional derivatives thereof.
 35. Theproteinaceous substance of claim 34, further comprising at least oneAcmA type domain with a relatively high calculated pI and at least-oneAcmA type domain with relatively lower calculated pI.
 36. Theproteinaceous substance of claim 34 or 35, wherein at least one domainoriginates from the AcmA type domain of the lactococcal cell wallhydrolase AcmA.
 37. The proteinaceous substance of any one of claims34-36, wherein at least one domain originates from the AcmA type domainof the lactococcal cell wall hydrolase AcmD.
 38. The proteinaceoussubstance of any one of claims 34-37, further comprising a reactivegroup.
 39. A nucleic acid molecule encoding the proteinaceous substanceof claim
 38. 40. A vector comprising the nucleic acid molecule of claim38.
 41. An expression system comprising the nucleic acid molecule ofclaim
 39. 42. An expression system comprising the vector of claim 40.43. An expression system capable of expressing the proteinaceoussubstance of any one of claims 34-38.
 44. An expression system of anyone of claims 41-43, wherein said expression system comprises amicroorganism.
 45. The proteinaceous substance of any one of claims34-38, further comprising cell-wall material.
 46. The proteinaceoussubstance of claim 38, wherein said cell-wall material is obtained froma method, said method comprising: treating said cell-wall material witha solution capable of removing a cell-wall component from said cell-wallmaterial, said cell-wall component selected from the group consisting ofa protein, a lipoteichoic acid, a carbohydrate, and mixtures thereof;wherein said cell-wall material has an improved capacity for bindingsaid proteinaceous substance.